JP2003514224A - Biosensing using surface plasmon resonance - Google Patents

Abstract

  (57) [Summary] The present invention provides methods and reagents for enhancing surface plasmon resonance (SPR) based detection assays. The methods and reagents can be used in any molecular recognition assay using a solid support. The present invention also provides an SPR device that works in an imaging mode.

Description

Detailed Description of the Invention

[0001] Technical Field The present invention generally concerns a surface plasmon resonance sensor. In particular, the invention relates to devices, methods and reagents for amplifying the surface plasmon resonance response and increasing its sensitivity, flexibility, throughput, specificity and range. BACKGROUND ART Surface plasmon resonance (SPR) is a general spectroscopic method for sensing a change in refractive index near the surface of a metal thin film. Its susceptibility to these changes provides a foothold that can be used to observe and quantify chemical reactions at the metal / solution interface, if the chemistry is well designed. The universality of this technology has led to its application to various chemical systems including biosensing (
Specific commercially available equipment is available).

SPR allows the detection of small changes in refractive index caused by interactions between surface-bound biomolecules and chemical species generated in the liquid phase. For example, immobilization of proteins on the sensor surface allows the detection of protein binding, which results in changes in the refractive index and thus in the angle-dependent reflectivity of thin metal films. This type of SPR sensing is typically performed in a commercial device using a carboxylated dextran gel on an Au film as the sensor surface, where the gel acts as a host for a surface bound binding partner. ing. However, SPR has also been applied in many other ways, including imaging SPR that can rapidly query multiple chemistries simultaneously. Similarly, various other surface chemistries have been used as a foothold for measuring biomolecular interactions.

SPR is 50 n of Au, Ag, Al or Cu anchored to a glass support by a thin adhesive layer of a free electron metal (eg Ti, Cr or mercaptosilane).
m thick film) relies on optical excitation of surface modes. Back-side, p-polarized irradiation of the Pumriz-bonded film at an angle greater than the critical angle of total internal reflection results in plasmon excitation at the metal-solution interface. Plasmon excitation is observed as an increase in optical absorption (decrease in refractive index) at the optimum coupling angle. This in turn gives rise to a local minimum in the SPR profile (reflectivity vs. angle plot), which is termed the plasmon angle (θ _p ). Sensing by SPR is possible because of the sensitivity of θ _{p to} changes in the refractive index near the metal surface. Adsorption, desorption and molecular-molecular interactions that occur at the metal-solution interface cause such changes, thereby inducing a shift in the plasmon angle. These changes can be monitored in real time, making SPR suitable for dynamic sensing.

Perhaps the most widely studied area of chemistry that uses SPR is protein-protein interactions, and signal transduction due to binding occurring is difficult or impossible to achieve with conventional optical spectroscopy. It is possible. To reduce non-specific binding and to increase surface loading of biomolecules, SPR experiments performed on commercially available devices usually use extended coupling matrices in connection with surface sensing. Such measurements typically begin with a single protein immobilized on a labeled substrate containing a carboxylated dextran (or "extended coupling") matrix layered on top of a thin vapor deposited Au film (ie, , Between the film and the sample). The result is a stretched three-dimensional array extending approximately 200 nm away from the surface of the Au film. Detected through corresponding small changes in total refraction provided.Detection of small molecules (<1000 MW) is still a daunting task for commercial devices, despite the signal amplification provided by the dextran matrix. In the case of, even the detection of species in the 2,000-10,000 MW range can prove to be difficult.

The use of coupling matrices is associated with a number of additional drawbacks, including non-specific interactions with signal-directing biomolecules and elimination of large molecules. Moreover, improper orientation of biomolecules within the matrix often leads to low activity of biomolecules (especially proteins). Since mass transport to molecules immobilized in the matrix is usually pH-dependent, another step is required during the assay for optimal diffusion of reagents into the matrix. These shortcomings have led to significant difficulties in assay design. It would be desirable to avoid these problems by using a planar SPR substrate such as an Au film modified with a monolayer of a bifunctional organic crosslinker (ie, without an overlying matrix). Let's do it. However, the SPR reflectance changes within these more homogenous substrates are often very small to measure in actual assays.

The applicability and effectiveness of SPR can be significantly extended if the ligand binding causes a pronounced change in refractive index, and thus a noticeable shift in plasmon angle. With such high sensitivity, the present technology can be widely applied to high throughput screening of low molecular weight drug candidates.

Devices, methods and reagents for SPR refractive index change amplification in chemical assays, particularly biomolecular recognition assays on planar SPR substrates coated with a monolayer or submonolayer of capture reagents (eg, antibodies), have been developed. It is an object of the invention to provide. It is also an object of the present invention to provide methods and reagents for ultrasensitive, non-PCR based DNA detection assays. Furthermore, with a resolution of 3-A, 1 to 5
It is also an object of the present invention to describe an SPR-based method for measuring the distance between surface constrained bodies on the 00 nm scale.

It is also an additional object of the present invention to allow high throughput screening reactions (both primary and secondary) with amplified imaging SPR. Replace (or instead of) the awkward deposition methods used in the field
It is yet another object of the present invention to provide a "wet chemistry" method for Au film synthesis for SPR (or other surface spectroscopy).

[0009] Finally, it is an object of the present invention to provide an imaging SPR device capable of depicting the spatial difference in film reflectance at a fixed angle of incidence, where the spatial difference is different refractive index or film thickness. (Ie different chemical modifications). The imaging SPR device provided by the present invention allows the use of SPR in a multiplex biological "chip" assay format. For example, an imaging SPR device would be essential for the simultaneous detection of multiple target analytes using a solid support in which ligands for different target analytes are bound to specific locations.

The devices, methods and reagents of the present invention enable unprecedented sensitivity and selectivity of chemical assays (multiplex biosensing assays). SUMMARY OF THE INVENTION The present invention provides devices, methods and reagents for SPR index change amplification. In one series of embodiments, colloidal-metal nanoparticles are used as optical labels for SPR-based sensing assays. These aspects rely on the observation that the SPR response of metal films is dramatically altered by the localization of such colloidal-metal nanoparticles to the film surface. The dramatic change in SPR response on the film surface caused by the adsorption of colloidal metal nanoparticles is caused by the occurrence of molecular recognition (
For example, it can be utilized in assays that rely on the binding of an antigen to an antibody, the binding of a ligand to its receptor, or the hybridization of complementary nucleic acid molecules. In one of many possible ways, one molecule involved in molecular recognition is immobilized on (or completely bound to) the metal film surface of the SPR substrate. Another molecule involved in the interaction with the immobilization molecule-by directly binding to the immobilization molecule or by binding to a third molecule, which in turn directly binds to the immobilization molecule. -Is labeled with colloidal metal nanoparticles. Bonding between the involved molecules leads to colloidal metal adsorption, with accompanying changes in the SPR response of the film. Methods for producing monodisperse colloidal metal nanoparticles in solution are well known in the art and are methods for attaching biomolecules to metal nanoparticles without loss of biological activity. In some embodiments, the use of colloidal Au nanoparticles has led to a 100,000-fold increase in SPR sensitivity.

For example, colloidal Au nanoparticles can easily bind to the ligand of the target analyte.
Then, when the target analyte begins to localize to the surface of the SPR film (or the ligand immobilized on it), the Au-labeled ligand is brought to the surface, and when the ligand binds to the target analyte, the SPR reflection Leads to an amplified change in the rate. Signal amplification is large enough to allow the use of planar SPR substrates (such as Au films modified with bifunctional organic crosslinkers), thus avoiding some of the problems associated with commercially available dextran-based matrices. I'm avoiding it.

Dextran-based SP despite significant limitations as discussed above
The R substrate is sufficient for some types of assays. Thus, the present invention also provides a method for SPR reflectivity amplification within a dextran-based matrix. In some embodiments of the invention, the Au nanoparticles attached to the ligand are first brought to a matrix and retained there through the interaction of the biomolecules underlying the assay. The nanoparticles are chosen to be small enough in diameter to allow efficient diffusion into the network. However, nanoparticles of this size do not optimally amplify the change in SPR reflectance. As a result, the nanoparticles are enlarged by the addition of Ag plating solution in order to optimize the amplification.

In another aspect, the present invention provides a method for further enhancing colloidal Au-amplified SPR reflectance by layering an inorganic film on the Au surface. The distance between the metallic nanoparticles and the metallic surface has a significant influence on the SPR reflectivity. In a preferred embodiment, a 30 nm thick film of SiO ₂ is deposited on a 50 nm Au surface. When colloidal gold nanoparticles were attached to the surface - either directly or, for example, a protein - a protein interaction -, the SiO ₂ layer to optimize the coupling distance between Au nanoparticles and Au surface It works as a spacer. This leads to a larger shift in the plasmon angle (ie the angle of minimum reflectivity) than observed using colloidal Au nanoparticles and Au surface without SiO ₂ film. This enhancement is observed even when the Au surface is bound to very low levels of colloidal Au (eg surface coverage less than 1%).

In another series of embodiments, the method is used to provide a “sandwich” immunoassay. In particular, the primary "capture" antibody to the target analyte is immobilized on the surface of the SPR substrate, either on the Au surface itself or on a layered SiO ₂ film (or other substance). The secondary antibody to the target analyte is conjugated to the colloidal Au nanoparticles by one of various methods. The Au nanoparticles and SPR substrate are then contacted with a biological fluid suspected of containing the target analyte. If present, the target analyte becomes bound to the SPR substrate through interaction with the immobilized capture antibody, and the colloidal Au localizes to SPR through the interaction between the secondary antibody and bound target donor. Will be changed. As a result, an amplified change in plasmon angle becomes observable, amplified with respect to the angular shift observed when the secondary antibody is not conjugated to colloidal Au nanoparticles. In some cases, the method provides 100,000-fold or more SPR signal enhancement compared to a conventional SPR assay using a dextran-based matrix,
It provides the ability to detect protein concentrations below 70 fM. It will be apparent to those skilled in the art that such an assay can be performed in a separate step, where the analyte-containing solution is first exposed to the surface and a washing step is performed to remove non-specific binding. Next, in the second step, the colloidal Au: antibody complex is introduced.

In another series of embodiments, the method of the invention is applied to detect a target nucleic acid sequence. For example, a "first nucleic acid probe" is immobilized on the surface of a planar SPR substrate, and a "second nucleic acid probe" is conjugated to colloidal Au nanoparticles.
The probes have regions that are at least partially complementary (or capable of some other interaction) with the target nucleic acid sequence such that both probes become associated in the presence of the target sequence. As a result, the colloidal Au nanoparticles conjugated to the second nucleic acid probe become localized to the SPR substrate, and there is an amplified plasmon angle shift. The method of the present invention has a dynamic range of about 5 digits and a 24-me
Allows a quantitation limit of better than 8 × 10 ⁷ molecules / cm ² for r oligonucleotides. This is an improvement of over 1000 times the sensitivity observed without using the amplification method of the present invention. In addition, in this embodiment, the introduction of the second nucleic acid probe may be before, simultaneously with, or after the introduction of the target sequence.

The present invention also provides an SPR device that can work in an imaging mode rather than a scanning mode. The scanning modal SPR is a single SPR for all SPR substrates at each angle of incidence (and / or wavelength of incident light) within a specified range.
Measure reflectance. Imaging modalities use a charge coupled device (CCD) to provide an image depicting the local SPR reflectance through an SPR substrate at one or more pre-determined angles of incidence. The angle of incidence selected is the angle at which the greatest change in SPR reflectance is observed due to the occurrence of an event that is assayed for SPR reflectance (eg, due to the binding of immobilized ligand to its target). . This allows SPR to be used as the basis for detection in a microarray format for the purposes of multiplex assays. In such an assay, the SPR device uses SPR after addition of a liquid suspected of containing labeled analyte.
Capture a normalized reflectivity image of the substrate. In the image obtained, the reflectivity signal intensity at each "spot" on the substrate gives information about the amount of target analyte present at that location. The imaging SPR device described herein allows easy introduction of large quantities of sample (using commercially available optics up to 50 mm x 50 mm microarray substrate). In addition, custom optics can use larger surfaces (eg, 6 inches by 6 inches). Unlike previously described SPR devices, the imaging device of the present invention provides a unique horizontal sample geometry, which allows robotic manipulation to regulate substrate handling and liquid delivery. This geometry avoids the need to flush the solution onto a vertical SPR substrate as was required with prior art devices.

Modifications to particles and / or surfaces are aimed at increasing the sensitivity and selectivity of SPR and include the use of: (i) Wavelength-dependence of amphoteric coupling to the surface. AuS / Au core designed to optimize sexual components-
New types of metal nanoparticles such as shell particles; (ii) particles that exhibit reduced non-specific binding and have sophisticated surface functionalization that allows for the regulation of the number of biomolecules bound to the particles; (iii) ) Substrates with subwavelength holes designed to provide local field enhancement;
And (iv) an oxide-coated metal film that optimizes the distance-dependent component of the particle-surface coupling. Instrumental efforts to increase sensitivity include the use of tuned excitation wavelengths, angle-dependent surface plasmon scattering measurements parallel to SPR, and interferometric SPR. A third aspect procedure for improving SPR sensitivity involves the use of two secondary signal amplification schemes. One is based on particle "growth", where selective chemistry is used to grow the size of immobilized particles; the second is based on the cascade effect, where one metal nanoparticle is used. Molecular recognition-inducible binding of particles leads to selective deposition from solution by some others.

To define the boundaries of SPR substrate regions for different chemistries, one method has been developed for making disposable replicas of printed circuit boards. The resulting "nano-beaker" (finished from polydimethylsiloxane or PDMS)
Is a small surface that allows to individually treat many spatially distinct regions. The use of such a microwell assay illustrates that changes in monolayer Au particle coverage of less than 0.1% are easily detected by imaging SPR. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a number of detailed protocols for ultrasensitive biosensing assays enabled by this generalized sensing scheme. However, to aid in understanding the present invention, a detailed description of the SPR response enhancement observed on colloidal metal adsorption (via molecular interaction) on SPR-based metal films is also provided. The following definitions are also provided: "SPR substrate" refers to any substance in which surface plasmon resonance phenomena (ie, optical excitation of surface modes (plasmons) in free electron metals when coupled to a prism) can be observed. Means In a preferred embodiment, the SPR substrate comprises a free electron metal film deposited or plated on the glass surface. Free electron metals include, but are not limited to, Au, Ag, Al and Cu. The present invention also contemplates the use of alloys or mixtures of metals.

By “SPR reflectivity” (or “reflectance”) is meant the modulation of the light intensity reflected from a prism to which an SPR substrate is bonded. SPR reflectivity is minimal when surface plasmons are excited on the SPR substrate. The SPR reflectivity value can range from 0% to 100% of the initial intensity of incident illuminance.

By “SPR profile” is meant the SPR reflectivity value of an SPR substrate at various angles of incidence (or at various wavelengths of incident light). SPR profiles are often plotted as SPR reflectivity versus angle of incidence (or versus wavelength of incident light). The "incident angle" means the angle at which the prism surface to which the SPR substrate is bonded is encountered when light enters the prism.

A “prism” is an optical element associated with an SPR substrate through which incident light is directed, eg, polished SF11 glass, on which is deposited an Au film. Preferred prisms of the present invention include triangular prisms (most preferably having a 70 ° -70 ° -40 ° geometry) and hemispherical prisms.

Although many examples and aspects herein describe the use of Au films and colloidal Au nanoparticles, other metals with SPR response, including alloys and mixtures of metals, are also available. It should be understood that it is contemplated and within the scope of the present invention. Similarly, although the embodiments of the methods of the invention described herein are often directed to biosensing assays, the methods are also applicable to other SPR-based sensing formats.

An important feature of the present invention is the ability of multiplex assays using the SPR technology of the present invention. Spatial multiplexing (ie arraying) can be enabled by physical separation of reagents on a metal substrate. However, additional multiplexing can be achieved by changing the properties of the metal nanoparticles. Changes include changes in the size and composition of the metal colloidal nanoparticles. Such multiplexing is best investigated using conventional SPR, and the reflectivities of the films (as well as the scatter from them) can be easily measured at all angles of incidence. However, multiplexing can be done by concatenating both light reflectance and scattering if possible, and ideally after measuring reflectance as a function of angle of incidence,
It can be performed using imaging SPR.

FIG. 1 shows the change in SPR response with colloidal Au adsorption on Au film. 50n to plot the SPR profile (reflectance as a function of angle of incidence)
The m-thick Au film is a bifunctional organic linker, 2-mercaptoethylamine (ME
Modified with A). The thiol groups of MEA are attached to the Au film, leaving free amino groups exposed on the surface. The solid line shows the sharp plasmon minimum (minimum reflectance value) at an angle of 43.7 °. Au film is 11 nm-diameter colloidal A
When exposed to a 17 nM solution of u for 1 minute, the Au particles bind to the Au film through these free amine groups. As indicated by the dotted line in FIG. 1, this results in a significant increase in minimum reflectance value, broadening of the SPR profile, and zero plasmon angle.
． A shift of 2 ° is produced.

It was observed that longer exposure of the Au film to the colloidal Au nanoparticles increased the extent of these changes. Thus, 60 minutes exposure shifted the plasmon angle by 2.6 ° (ie to 46.3 °) and localized colloidal A
The minimum reflectance value of Au film without u nanoparticles ("naked" Au film) increased by almost 40%. A more complete discussion of the materials and methods used to generate the SPR profile of FIG. 1 is provided in Example 1.

These results resemble the dramatic shifts induced by thin carbon coatings deposited on Ag surfaces, where the strong adsorption of the film induces a significant degree of plasmon damping. I don't want to be bound by a single theory or mechanism,
The SPR change due to u-colloid adsorption appears to be due to a similar adsorption decay process, as these particles have a non-zero imaginary dielectric component. The second possible (but not exclusive) mechanism of decay is a situation similar to the effect observed with roughened Au films and other grain-modified films, where localized surface plasmons of colloids and Au films Includes coupling with propagating plasmons.

When the time course of reflectance change is monitored at minimum reflectance,> 1% reflectance change is readily observed in the first 100 ms of colloid adsorption. The surface coverage at this point is approximately 0.1% of the densely packed monolayer. If the plasmon angle changes linearly with the surface coverage (a reasonable assumption for low coverage) and the device angular resolution limit is 0.005 °, then 40 times the surface coverage ( A decrease of 0.2 ° / 0.005 ° is detectable. Therefore, 0.001 of 12 nm-diameter colloidal Au monolayer
25% (2.0 × 10 ⁷ particles / cm ² ) can be easily observed. As discussed below, even lower coverage is observable using larger diameter Au colloids. The time course of reflectance as a function of time is shown inset in FIG.

The effect of colloidal Au adsorption on the SPR response of Au film substrates is dramatically dependent on the size of colloidal nanoparticles. The solid line in FIG. 2 is coated with MEA, followed by the same number of densities (1.3 ± 0.15 × 10 ⁹ particles / cm ² ) 30, 35,
Figure 5 shows the SPR response of 47nm thick Au films coated with 45 and 59nm diameter colloidal Au. Despite a relatively low number of densities, the SPR profile varies dramatically with particle size, with a minimum reflectance of 10% for diameter increases of less than two-fold.
From 35% to 35%. In fact, at the largest particle size (59 nm), SPR
The profile closely resembles that of the bare evaporated film; the profile is shallow and broad, without the well-defined plasmon minimum typically observed in SPR. A more complete discussion of the materials and methods used to generate the SPR profile of FIG. 2 is provided in Example 1.

Although the number of colloidal Au particles per unit area was kept constant in the data shown in FIG. 2, it is important to note that the split surface coverage varies with particle size. . In other words, an equal number of two different sized particles will cover different geometric regions. Taking this into account, the split coverage of 30, 35, 45 and 59 nm diameter Au particles is 1.2, respectively for densely packed monolayers.
1.6, 2.6 and 4.5%.

FIG. 3 shows the changes that occurred in the SPR profile for surface concentration (expressed in number of particles / cm ² ) for 35 nm (FIG. 3A) and 45 nm (FIG. 3B) diameter colloidal Au particles. .. For 35 nm particles, a low number of particle densities induces relatively small plasmon angle shifts and reflectance changes. As the number density increased, the change in position and degree of minimum reflectivity increased, eventually reaching a total plasmon shift of about 7 ° and a reflectance increase of> 40%. A somewhat larger angular shift is observed for 45 nm diameter colloidal Au particles; the first change in plasmon angle is significant and increases rapidly with increasing surface concentration.
However, the shape of the SPR profile evolves even more rapidly for 45 nm diameter particles, with> 30% reflectance observed at the lowest surface concentration. This trend was similar for other sizes of colloidal Au particles-smaller perturbations are observed for 30 nm diameter particles and larger initial reflectance changes are observed for 59 nm diameter particles.

From a practical point of view, it is not the overall perturbation of the profile that is important in the SPR sensing format, but the maximum observable change in plasmon angle shift or reflectance (
Increase or decrease). From the data presented in Figures 2 and 3, it is clear that at higher surface coverages, the SPR profile is rather too broad to determine the exact plasmon angle. Therefore, in a preferred embodiment, both from a device and sensitivity point of view, the reflectance change itself does not have a clear physical meaning in terms of the dielectric properties of the interface, despite the fact that at a single angle. It is more practical to measure reflectance changes.

In a preferred embodiment, the angle selected to measure reflectance is the angle that causes the maximum reflectance change upon binding of colloidal Au nanoparticles to the Au film. Maximum reflectance change is SPR of Au colloid-modified film and unmodified film
It can be determined by considering the difference between the profiles. FIG. 4 shows the difference profile calculated from the data shown in FIG. A maximum increase in reflectance was observed at 43.7 ° for both the 35 nm diameter particles (Figure 4A) and the 45 nm diameter particles (Figure 4B). This result suggests that the minimum of the SPR profile of the unmodified film is the optimal viewing angle for the Au colloid-modified film. The large size reflectance changes observed here suggest that lower surface concentrations are observable. In fact, for 45 nm diameter particles, 1.3 × 10 ⁹ particles / c
A number density of m ² gives a reflectance change of 40%. Current scanning SPR devices are <0.
Assuming a linear SPR response at low coverage, it is possible to detect surface coverage that is 1/400 lower (3x10 ⁶ particles / c), since a 1% reflectance change can be detected.
m ² , corresponding to 0.0065% of the monolayer).

In FIG. 5, the maximum changes in reflectance are 30 nm, 35 nm, 45 nm and 5
Plotted as a function of number density for 9 nm diameter colloidal Au particles. For all particle diameters, the peak of the difference profile is about 43.7 °, again indicating that the ideal angles are the same regardless of particle size. For assay and sensing applications, this is a clear advantage of particle facilitation, as the angle at which the greatest signal will be observed can be known prior to performing the assay. In some cases this can eliminate the need for high resolution scans to determine the amount of immobilized material. Measuring percent reflectance at a particular angle is also less error-prone at higher surface coverage, as absolute plasmon angles are difficult to determine.

It should be noted that although the focus of the present invention is SPR, it also includes other analytical techniques used to aid characterization, which are themselves sensitive DN.
A detection can be provided. Thus, in the absence of significant non-specific particle binding, single nanoparticles are readily visualized by atomic force microscopy, field emission scanning electron microscopy or short range scanning optical microscopy. If the detection limit required for DNA cannot be achieved by SPR, other techniques are available. Biosensing Assay Format One assay format in which the methods of the invention can be readily used is the "sandwich" assay, which is well known in the art. The sandwich assay is generally organized as follows: ligand A immobilized on a solid support can bind to target analyte B, which can simultaneously bind ligand C. When contacted, a complex is formed between A, B and C on the solid support. If ligand C can be detected
If labeled with a "tag", then the amount of target analyte B can be quantified by measuring the signal of the detectable tag present on the solid support. Many variations on this basic scheme are possible. It will be understood that a "target analyte" is any analyte that is the subject of an assay; suitable targets of the present invention include, but are not limited to, nucleic acids, proteins, hormones, sugars and metabolites. A "ligand" is any species capable of binding to a target; suitable ligands of the present invention include, but are not limited to, antibodies and nucleic acids. The sandwich assay format is commonly used in immunoassays, where the "ligand" is an antibody directed against a target analyte.

FIG. 6 shows an example of a sandwich format in which the first antibody against the antigen is immobilized on Au film. A second antibody to the antigen is paired to the colloidal Au nanoparticles. When contacted, a complex is formed on the solid support between the first antibody, the antigen and the second antibody paired to the colloidal Au nanoparticles.

FIG. 7 shows data from an assay in the format shown in FIG. 6 using a planar SPR substrate (ie consisting of a conductive film without a laminating matrix).
To obtain this data, a 50 nm thick Au film is first modified with 3-mercaptopropionic acid (MPA), thereby forming a carboxylic acid terminated self-assembled monolayer (SAM). The SPR profile obtained with this modified film alone is shown by the solid line in FIG. 7A. Next, γ-chain-specific monoclonal goat anti-human I
The gG antibody [ah-IgG (γ)] ("primary antibody" or "primary antibody") was coupled to the carboxylated monolayer by carbodiimide coupling to the free amino portion of the antibody. Modified film and human IgG [h-IgG; 150 kD]
Incubation with a 0.09 mg / mL solution of (“target antigen”) produced a shift in plasmon angle of only 0.04 °. This is indicated by the dotted line in Figure 7A. In fact, the plasmon angle shift is so small that the antigen (h-I
The SPR profile in the presence of gG) almost overlaps with the SPR profile obtained from the modified film alone. Exposed to 8.4 mg / ml solution of the film more F _c specific monoclonal goat anti-human immunoglobulin G antibody _{[a-h-IgG (F c} ) ] ( "secondary antibody" or "secondary antibody") Even small (0.0
6 °) Only plasmon angle shift occurs. This is shown by the long dashed line in FIG. 7A. However, the secondary antibody [10] has an electrostatic pair conjugate [10 nm diameter Au particle].
a-h-IgG (F _c ) -Au], the plasmon angle of 1.
A 7 ° shift is observed, with a 2% increase in minimum reflectance and a detectable broadening of the SPR profile. This is shown by the long dashed line in FIG. 7B.
This colloid-induced 1.7 ° shift is the shift observed in the unamplified assay using the same planar substrate (ie, 0.06 ° as shown by the long dashed line in FIG. 7A).
28 times larger than Therefore, the use of protein-Au colloid pair conjugates in this biosensing embodiment provides a significant increase in sensitivity. Biochemical specificity is also maximized under these conditions -a-h-IgG (γ) modified surface is human serum (excluding IgG), human immunoglobulin A and [a-h- IgG (
F _c) -Au] exhibit no reactivity little or the. a-h-IgG (γ
) -H-IgG modified surface is also highly specific and is small (0.08% upon exposure to a 10 nm diameter Au pair conjugate with goat anti-human immunoglobulin G1 (a-h-IgG1-Au). °) Only leads to plasmon shift. A more complete discussion of the materials and methods used in this immunoassay is provided in Example 2.

As mentioned previously, in a preferred embodiment, the assay is performed by measuring the reflectance change in the SPR substrate at a previously determined angle of incidence. The optimum angle is the angle at which the largest change in reflectance is observed due to the occurrence of the biological event being detected. In the sandwich assay, this biological event is the formation of the primary antibody-antigen-secondary antibody "sandwich". To determine the optimum angle,
The differential SPR profile (change in reflectance with respect to the angle of incidence) is prepared by calculating the difference between the SPR profiles obtained with the secondary antibody and antigen steps. The peak of the difference profile occurs at the largest difference between the two spectra and therefore represents the optimum angle for monitoring reflectance changes between reactions.

FIG. 8 shows the differential SPR profile of the data provided in FIG. ah
-IgG (F _c) For the addition, the maximum reduction in the reflectance of only approximately 2% is obtained at an incident angle of 54.3 ° (long dotted FIG. 8). In contrast, a-h-IgG (F c) -Au
With addition, a -50% decrease in reflectance is observed at the same angle of incidence (solid line in Figure 8).
. Therefore, the 10 nm Au-paired secondary antibody leads to a 25-fold increase in sensitivity compared to the unpaired secondary antibody.

FIG. 9 shows the reflectance time course for immobilization of both unpaired and Au-paired secondary antibodies. When the reaction was monitored at an angle smaller than the plasmon angle (53.4 ° 50% reflectance), a reflectance change of> 40% was observed in the case of Au-paired secondary antibody (see “Antibody: Au in FIG. 9”). Immobilization of unpaired bound antibody on identically prepared surfaces, on the other hand, results in reflectance changes of <2% (line marked as "free antibody" in Figure 9). This illustrates the enhanced sensitivity in assays that rely on real-time monitoring of biospecific interactions. This enhancement is protein-A
It should be noted that u pair binding is obtained despite being diluted more than 6 orders of magnitude over the free antibody (17 nM vs 56 mM).

The utility of the particle enhancement methodologies of the present invention becomes most noticeable at low concentrations of antigen. FIG. 10 shows the change in colloidal Au-induced plasmon angle as a function of h-IgG concentration. The h-IgG concentrations used were 3 mM (Figure 10A), 0.3 mM (Figure 10B), 3.0 nM (Figure 10C) and 6.7 pM (Figure 10D). In each figure, the solid line shows the SPR profile of the ah-IgG (γ) modified film; the long dotted line shows the SPR profile of the ah-IgG (γ) modified film in the presence of h-IgG only. Is shown; the dotted lines are h-IgG and a-h
3 shows an ah-IgG (γ) modified film in the presence of both -IgG (F _c ) -Au. As expected, changing the solution antigen concentration over almost 6 orders of magnitude (from 3.0 μM to 6.7 pM) reduces the observed plasmon shift. Lowest h-IgG antigen concentration-3.0 nM (Figure 10C) and 6.7 pM (
Figure 10D) - In, a-h-IgG (F c) -Au can not be detected in the absence SP
Induce R change. Despite these low antigen concentrations, a marked shift due to particle immobilization is still apparent when 10 nm Au-paired secondary antibody is used. Even the lowest concentration of h-IgG tested (6.7 pM = 1.0 ng / m
l), an easily detectable shift of 0.33 ° (FIG. 10D). The ease with which this shift is analyzed indicates that the actual detection limit of the 10 nm Au versus bound secondary antibody is even below the picomolar range.

The immunosensing embodiments described above all use planar SPR substrates (
That is, an Au film modified with a monolayer of a bifunctional organic linker to which the primary antibody will be covalently attached). As discussed in the background section, commercially available SPR assays are typically used to extend capture carboxylated dextran matrix (eg 200-4) to concentrate capture molecules such as primary antibodies.
00 nm thick) coated Au film is used. Molecules to be localized on the film surface (eg, primary antibodies) are covalently bound by a dextran matrix, resulting in the formation of a three-dimensional array of molecules extending ~ 200 nm away from the surface. As outlined in the background art, there are many drawbacks to using such a matrix. The method of the present invention dramatically surpasses the sensitivity of such prior art. For example, the use of a 10 nm Au-paired secondary antibody on a planar SPR substrate gives a 3,000-fold limit of detection compared to the same assay performed with dextran matrix using an unpaired secondary antibody. Has been enhanced.

In some embodiments of the invention, colloidal Au having a diameter greater than 10 nm.
Even greater increases have been obtained with the use of nanoparticles. For example, if the immunoassays described above were performed using a 41 nm Au-pair-conjugated secondary antibody and a planar SPR substrate, a 30-fold sensitivity amplification would be obtained over the same assay using an 11 nm Au-pair-conjugated secondary antibody. can get. This represents a 100,000-fold increase in sensitivity when compared to a conventional SPR assay using a dextran matrix. In fact, 41 nm Au-pair bound secondary antibody and planar SP
With the R substrate, it is possible to detect h-IgG at concentrations as low as 10 pg / ml.

In some aspects of the invention, a conventional dextran-based matrix is used as a substrate for colloidal Au-stimulated SPR detection. However, depending on the matrix pore size, the colloidal Au particles used above will be excluded from the interior of the matrix by steric hindrance. Different methodologies are required for such embodiments: use colloidal Au nanoparticles that are small enough to enter the matrix and then, after they enter the matrix, Au ³⁺ and They are selectively made larger by the addition of NH ₂ OH, or a metal plating solution, preferably a commercially available Ag plating solution. By this method,
The u nanoparticles can be enlarged in the matrix by adding a plating solution to a size that significantly enhances the SPR response. For example, for the sandwich immunoassay, 1.8 nm colloidal Au can be paired to a secondary antibody. The resulting counter-conjugate can diffuse into the matrix, in which the primary antibody is covalently bound, along with the target analyte to be detected. Following preparation of the (primary antibody)-(target antigen)-(secondary antibody) complex in the matrix, the Ag plating solution is flown over the film to increase the colloid size. Using commercially available Ag plating solutions, it is possible to selectively enlarge paired Au nanoparticles without nucleation of new particles. In a preferred embodiment, the Au film substrate on which the dextran is overlaid is such that the Ag plating solution is directly exposed through the pores of the dextran matrix.
To prevent Ag plating of the u film, it is first pretreated with a short chain alkanethiol such as 6-mercaptohexanol. When this method is used to perform the h-IgG immunoassay, a 30-fold enhancement is obtained over the same assay performed with a 41 nm Au-paired binding secondary antibody. A 10 ⁶ fold enhancement is obtained when compared to the same dextran-based assay performed using an unpaired Au secondary antibody.

All of the above immunosensing embodiments are compatible with the detection of any target analyte for which suitable primary and secondary antibodies are available. In addition, the described method is applicable to any molecular recognition assay in which one of the related species in molecular recognition can be immobilized on a planar SPR substrate and another related species can pair-bond to colloidal Au nanoparticles. is there.
For example, the receptor-ligand interaction may be by immobilizing the receptor on a planar SPR substrate and then pair-binding the ligand directly to colloidal Au, or by providing a colloidal Au-paired antibody capable of binding the ligand. It can be assayed by using. Moreover, the method of the present invention allows Au-rather than interaction-driven adsorption.
It can be used to study the release of Au-colloid tagging reagents from the film.

The methods described herein provide two examples of SPR-based assays based on molecular recognition: (1) one of the molecules involved in recognition is derivatized, a planar SPR Au film. An assay that is immobilized on a substrate and another molecule involved in recognition is paired to colloidal Au of size 10-150 nm; or (2) one of the molecules involved in recognition is an Au film. Immobilized within the overlying dextran matrix, and another molecule involved in recognition, is a colloid of sufficiently small diameter to allow the paired reagents to diffuse into the dextran matrix. Paired to Au-like Au; Au nanoparticles are enlarged by addition of Ag plating solution following diffusion of the paired conjugate into the matrix Assay. Both methods provide unique methods of signal amplification, and the better method is best determined by evaluating some general criteria. In addition to the criteria set forth below, other criteria will be apparent to those skilled in the art in determining the best method for a particular purpose.

Most important is the size and nature of the capture molecule (eg “Ligand A” or “primary antibody”) and the analyte to be immobilized. Large molecules will be excluded from diffusion into dextran; thus, are unavailable in the most sensitive areas (near the surface). Similarly, if the sample is large, diffusion to the immobilized receptor is impossible. Dextran matrices are also impractical if a specific orientation of the capture reagent is required for receptor binding, as in DNA hybridization or membrane studies. Many protocols are known in the art to orient DNA, bilayers or proteins on a flat surface.

The concentration of the target molecule must also be considered. For h-IgG, low concentrations (100 ng / ml-100 pg / ml) are well suited to promote Au on planar films with 11-nm diameter Au colloids; and lower concentrations require larger colloids. . However, the latter species appear to be more susceptible to degeneration (
Not usually observed). It also increases the likelihood of multiple interactions between protein-bound particles and the surface. In contrast, the 1.8-nm diameter Au colloid to conjugate gives a protein to colloid ratio of 1: 1 (in most cases). This aids quantification and due to optimized exposure to the Ag plating solution, very low concentrations (<100p
(g / ml) can be detected. However, no further immunochemical manipulation is possible after Ag plating. Finally, it should be noted that both surfaces can be subjected to regeneration using standard techniques.

Larger changes in SPR angle shift are expected for particles that can be more efficiently coupled to surface plasmons in Au films. Therefore, one aspect of the invention relates to generating particles with tunable absorption spectra for use as SPR tags. Absorption of light by Au nanoparticles leads to particle-based surface plasmon excitation (size-dependent effect). Therefore, SPR
One simple way to tailor the absorption spectrum for a tag is to use differently sized Au nanoparticles. Two problems traditionally associated with larger colloidal Au nanoparticles (ie, those with diameters greater than 35 nm) were polydispersity and instability (compared to smaller particles). Since the stability of colloidal particles is known to decrease with increasing size, some of the instability problems could be solved through improved monodispersity. Furthermore, for applications where Au nanoparticles are reagents, it is essential to have "reagent grade" (ie, relatively pure) materials.

It is easy to understand that a larger change in reflectivity is observed with larger particles (perhaps per particle, the larger scattering and dielectric properties associated with binding with more Au metal). Probably due to both changes), no significant changes were expected with very small molecular Au clusters. Smaller particles are larger Au
While giving less dramatic shifts than observed with tags, their smaller "footprints" allow for a higher degree of surface labeling, allowing single particle binding to correspond to single biological binding. It is easier to place. An additional benefit of reduced particle tag size is improved diffusion of tagged biomolecules.

Another example of efficacy with colloidal Au reagents in biochemical SPR experiments involves binding and displacement of colloidal Au-tagged proteins. In an embodiment of this aspect of the invention, anti-biotin is covalently attached to a planar SPR substrate. Introduction of biotinylated colloidal Au (produced by adsorption of biotinylated BSA onto 10-nm Au) led to small changes in reflectivity indicative of low coverage of the paired conjugate, which in turn resulted in small amounts on the surface. Active anti-biotin is shown. Antibody: Antigen
When the Au complex was exposed to high concentrations of biocytin, a water-soluble analogue of biotin that binds anti-biotin, the colloidal Au-tagged biotin was displaced and the SPR
Reflexivity returns to its original position. These experiments show that colloidal A on the SPR surface
It has been shown that the binding of the u-pair conjugate is reversible, allowing the use of SPR to detect binding of low MW species due to ligand displacement.

Using standard IgG immunoassays as model system, Au-particle amplification is SP
Although it has been shown to lead to enormous improvements in R sensitivity, this technique has been applied to several other systems, for example to detect soluble factors of biological importance in serum; For example, the cytokine interleukin-5 (IL-5). IL
-5 is produced by T lymphocytes and appears to be the major cytokine involved in the regulation of eosinophilia. Since eosinophils are important for allergies, IL-5 is believed to have a key importance in the development of allergic disorders (eg asthma). These molecules are good targets for biosensing because IL-5 and other cytokines are pharmaceutically important. The selectivity of the IL-5: Au pair conjugate for Protein A / anti-IL-5 on Au film is shown in FIG. The observed angular shifts were much greater for specific interactions than for non-specific interactions (ST4: Au vs. conjugates). Small amounts of non-specific binding (0.1 °) for ST4: Au. Met. The specific interaction was shifted a minimum of 1.6 ° (0.5% Blotto was used in these experiments to block non-specific binding). Although this experiment illustrates both the Au-amplification sensitivity and the selectivity of this assay, the biological sample will contain free cytokine, cytokine: Au vs. conjugate. A pseudo-competitive assay is used to detect IL-5 in such samples. In this experiment, protein A / anti-I
Since L-5 coated Au films mimic serum, they are first exposed to free IL-5 in the presence of high concentrations of human serum albumin. After collecting SPR curves, the surface was IL-
5: Au exposed to conjugate. Due to the slow dissociation kinetics of the anti-IL-5 / IL-5 interaction, exposure to paired conjugates produced little, if any, IL-5.
Only is displaced from the surface, rather IL-5: Au binds only to "open" antibodies on the surface. Therefore, the amount of angular shift caused by exposure to the pair conjugate is inversely proportional to the amount of analyte in the original (“serum”) sample.

In a preferred embodiment of the invention, further enhancement of the colloidal Au-paired SPR response is obtained by optimizing the coupling distance between Au nanoparticles and Au film. In some embodiments, inorganic spacers, preferably SiO ₂ deposited by thermal evaporation, are placed on the surface of the Au film to increase the distance between the colloidal Au and the Au film. In another aspect, optimization of the coupling distance is achieved by layering one or more polymer layers, preferably polystyrene sulfonate and / or polyallylamine hydrochloride, stacked on the Au film by electrostatic interaction. It is achieved by stacking. In a particularly preferred embodiment, a 30-40 nm thick SiO ₂ layer is used with 12 nm colloidal Au. FIG. 11 shows that the Au film is 15 nm (FIG. 11A) and 24 nm (FIG. 11B).
) And 33 nm (FIG. 11C) thick SiO ₂ layers and have 12 nm colloidal Au on them, showing progressive enhancement of SPR response.
In each figure, line A is the SPR profile of an Au film without a SiO ₂ layer or localized Au colloid; line B is the SPR profile of an Au film with only a SiO ₂ layer; line C is SiO ₂ 3 is an SPR profile of an Au film with layers and Au colloid deposited. The thickness of the spacer layer product is (i) A
u film thickness; (ii) film composition (eg, Ag film may give different results); (iii) excitation wavelength; (iii) colloidal Au.
The size of the particles; (iv) the composition of the colloid (for example, different results may be obtained with colloidal Ag); and (v) depending on the surface coverage, to some amount of immobilized colloid. It is understood that it produces the maximum shift. in addition,
There are many other materials that can be suitable spacers, including (but not limited to) metal oxides, sulfides, nitrides or any other material containing metal ions in the positive oxidation state. It will be understood.

In the SiO ₂ layer stack, a 30 nm thickness improves the sensitivity by a factor of 3. Similar results have been observed with polymeric spacer layers consisting of different cationic and anionic polymers. Not only does this development lead to improved detection limits, but it also extends the possibilities to all attachment chemistries used on glass surfaces, which can improve film stability.

The SiO ₂ coating (thin and arguably somewhat porous) also serves to protect the underlying metal from chemical attack. Reactivity is not relevant for Au films, but oxidation will preclude the use of Ag metal in SPR. Ag films are known to have sharper angle-dependent reflectivity curves, so would be a better choice without their rapid oxidation.

It would be an important property of the present invention that the distance of colloidal nanoparticles from the metal film could be essentially measured by SPR detection. Due to this property, the assay performed on the film surface does not need to require the presence or absence of colloidal nanoparticles. Rather, according to the invention, it is possible to carry out an assay in which the presence of an analyte acts to increase or decrease the distance of the colloidal nanoparticles from the metal film surface.

A preferred embodiment of the present invention uses colloidal Au nanoparticles to enhance the SPR profile of metallic SPR substrates. However, the metal nanoparticles are Ag, C
It is understood that other materials with complex refractive indices may be included, including (but not limited to) u, Al or alloys of two or more Au, Al, Ag and Cu. Should do. In another embodiment, the metal nanoparticles are Ag, Al, Au substantially coated with a shell of any metal, any oxide, any sulfide, any phosphide, or any organic or inorganic polymer. Or C
It consists of a core of u (or an alloy of two or more of these metals). In addition, although the preferred embodiment uses substantially spherical metal nanoparticles, it should be understood that other shapes are also contemplated.

A wide range of wavelength “tunability” is possible with AuS core / Au shell particles, which is Hau
s and Halas group. For example, Avertit
et al. , Phys. Rev. Lett 78: 4217-4220 (1
997). By changing the manufacturing conditions, it is possible to adjust the position of the second λ _max (adjustment index of the size ratio of the core to the shell). FIG.
Shows the optical spectra for several batches of these particles. 63
Particles that have a significant absorption at 2 nm are especially preferred as they are the most commonly used laser wavelengths in SPR studies. The strong absorption of surface plasmons at this wavelength results in a high degree of film-nanoparticle coupling, which in turn leads to a greater SPR response and enhanced sensitivity.

Polydispersity in particle production causes difficulties in describing SPR data because amplification is a function of particle size. Synthesis of AuS / Au shell particles by methods similar to those used for colloidal Au (ie, strict cleanliness, ultrafiltration solvent, rapid reagent addition, rapid stirring and reflux) was developed to monodispersity. Leads to significant improvements. A quantitative measurement of the extinction coefficient was performed in solution and on the surface for the obtained AuS / Au particles. On the surface, particle coverage was unambiguously established by AFM and FE-SEM. In solution, S and Au atomic absorption was performed on solution samples prepared such that all particles had been removed by aggregation / centrifugation. This makes it possible to determine if 100% of the added Au has been converted into dispersible particles. Subsequently, a combination of TEM image analysis and static light scattering is used to establish average particle size and dispersibility. These numbers, in turn, can be used to calculate the particle concentration in solution. For both sets of measurements, the observed data are correlated with theory using Bohren's program for calculating core-shell absorption and scattering.

To determine the optimal conditions for core / shell particles in ultrasensitive SPR applications, SPR spectra are collected at multiple wavelengths and the results are compared with the expected behavior based on the optical spectra of the particles. Of course, at each wavelength different Au film thickness and different collection angles are optimized; the relationship between these parameters is Fresne
It can be modeled very efficiently using the available software given by the equation l. Ultrasensitive SPR Detection of DNA Hybridization In certain preferred embodiments, the colloidal Au-enhanced method of the invention is used to detect DNA hybridization. In one aspect of the invention,
The solution suspected of containing the target DNA sequence will not colloid Au into the nucleic acid in the solution.
Is treated with a reagent that couples with. After pairing, the solution of nucleic acid is modified with an oligonucleotide probe having a sequence complementary to the target DNA sequence, the plane S
Contact with PRAu film substrate. If the target is present, it hybridizes to the immobilized probe, thereby binding the colloidal Au to the surface of the Au film. Changes that occur in the SPR response of the film can be monitored and the presence of target detected. Methods for attaching nucleic acids to metal films are well known in the art. For example, an amine-modified nucleic acid has a 1-ethyl-3- (3
It can be attached to a 16-mercaptohexadecanoic acid-derivatized surface via a (dimethylaminopropyl) carbodiimide / N-hydroxysulfosuccinimide bridge. A
Methods for DNA attachment to u-film are described by Herne et al. J. Am
． Chem. Soc. 119: 8916-8920 (1997) (incorporated herein by reference in its entirety).

In some preferred embodiments, a “sandwich assay” -type hybridization format is used for DNA detection; an example is shown graphically in FIG. In this example, an oligonucleotide probe (S1) having a sequence complementary to the first part of the target DNA sequence (S2) is immobilized on the surface of a planar SPR substrate. The SPR substrate is then treated with a solution suspected of containing the target DNA sequence (S2) and also with a colloidal Au-pair binding oligonucleotide having a sequence complementary to the second portion of the target sequence ( S3: Contact with Au). S1 and S3
The sequence of: Au is designed such that the sequences S1, S2 and S3: Au form a triplex hybridization complex in the presence of the target (S2). By this method,
Colloidal Au is brought to the surface of the Au film only if the target sequence (S2) is present. The resulting change in SPR response of the film can be monitored to detect the presence of the target sequence.

Colloidal Au can be prepared from oligonucleotides (S) by many methods known in the art.
It can be paired to 3). For example, thiol-labeled DNA can be produced, and the thiol group is M
irkin et al. , Nature 382: 607-609 (1996).
), Elghian et al. , Science 277: 1078-
1081 (1997), Storhoff el al. J. Am. Chem
． Soc. 120: 1959-1964 (1998), Storhoff an.
d Mirkin, Chem. Rev. 99: 1849-1862 (1999)
And Mucic et al. J. Am. Chem. Soc. 120: 12
674-12675 (1998), each of which is incorporated herein by reference in its entirety, and may serve to couple DNA to Au as described in US Pat. Alternatively, biotinylated DNA can be prepared and subsequently ligated into a streptavidin: Au pair conjugate. Example 3 provides a detailed protocol for making such paired conjugates. In some embodiments, the SPR substrate is treated with mercaptohexanol, which helps to "stand up" the immobilized probe DNA by reducing physisorption.

FIG. 13 shows an example of an SPR profile obtained using the above assay format. In this example, the target (S2) is a 24-mer oligonucleotide. S1 and S3: Au are 12mer probe oligonucleotides,
Each of them is complementary to one of the halves of S2. S1 has a 5'amine group through which it is immobilized on the Au film as described above. S3 has a 3'thiol group to which colloidal Au is attached. S1, S2 and S3 have the following sequences: 5'-H ₂ NC ₆ H ₁₂ -CGC ATT CAG GAT-3 'S1 5'-TAC CAG TTG AGA ATC CTG AAT GCG-3' S2 5'- line a of _{TCT CAA CTC GTA-C 6 H} 12 -SH-3 'S3 Fig. 13 shows the SPR response S1 pairing Au film under other reagents absent. Line B shows that hybridization of S1 to S2 in the absence of S3: Au leads to very small substitutions (0.1 °) with minimal SPR reflectivity. Line C shows that there is a pronounced plasmon angle shift when the S3: Au pair conjugate is added, which shows an approximately 18-fold increased SPR compared to the response observed in the absence of S3: Au. Corresponds to the response.

In certain preferred embodiments, the Au film is capped with spacers to optimize the coupling distance between the Au film and the colloidal Au, as described above. The preferred spacer is a SiO ₂ layer.

It will be appreciated that the application of Au-colloid SPR-enhancing methodologies need not be limited to DNA detection assays in the probe form described above. Two core elements:
(1) An immobilized probe whose sequence is complementary to a part of the target DNA sequence of interest (
Or probes); and (2) a number of assay formats for the detection of specific DNA sequences that rely on a detectable tag pair-bound to a molecule that will directly or indirectly associate with an immobilized probe. Is known in the art to exist. It will be appreciated that one of skill in the art can readily adapt the methods of the invention to enable SPR detection of target nucleic acid sequences in any assay format that relies on these two elements. Furthermore, the same methodology is applicable to forms in which the probe and / or target consist of other nucleic acids (eg RNA), nucleic acid analogs (eg peptide nucleic acids) or combinations thereof.

One of the unique properties of SPR-based assays is that binding data can be acquired in real time to monitor the progress of the reaction. The inset of FIG. 13 is
Reaction of the second hybridization step (ie, probe: Au vs. conjugate binding to surface-constrained oligonucleotides) by monitoring SPR reflectivity at a fixed angle of incidence (53.2 °) as a function of time. A kinetic plot is shown. As expected, the signal change is dramatic in the first 5 minutes and the hybridization process is almost complete after 60 minutes.

Changes in the SPR response of Au films can be reversed by DNA “melting” (ie, dehybridization by heating the substrate), or by DNA digestion of the triplex complex at the site of releasing Au nanoparticles. . Again, melting and DNA digestion can be monitored in real time to obtain kinetic data. In the case of DNA melting studies, the exact temperature changes in metal films induced by pulsed or continuous laser irradiation can be calculated exactly. The detection of single nucleotide polymorphisms (SNPs), the hybridization / dehybridization thermodynamics and kinetics thereof, are highly temperature sensitive, which is a precise laser-on Au film surface.
This can be achieved by monitoring the SPR response of Au film in response to induced temperature changes. In the case of DNA digestion, cleavage of the surface-constrained oligonucleotide / probe: Au complex-single-stranded or double-stranded region-can be observed as a shift in the plasmon angle as bound colloidal gold is released. Ah

As discussed above, the method of the present invention using scanning SPR allows detection at less than 3 × 10 ⁶ particles / cm ² for 45 nm diameter Au-colloid nanoparticles. If each particle is pair-bonded to a nucleic acid probe as described above, 3x1
It is possible to detect 0 ⁶ target nucleic acid copies / cm ² . DNA hybridization assays are described in Bowtel, Nat. Genet. Suppl. Two one
: 20-24 (1999) (incorporated herein by reference in its entirety).
, And is typically performed in a microarray format using reagent spots of approximately 50 μm diameter. Assuming a spot size of 50 μm, it means that the Au-colloid SPR-enhancing method provided herein can detect as few as 60 copies of target nucleic acid. Lower levels can be expected using larger Au colloidal nanoparticles with> 45 nm diameter. This level of sensitivity is unprecedented in the field. The methods provided herein offer new opportunities for PCR-less hybridization biosensors and general DNA diagnostics. Imaging SPR and Microarray Assays The technology of imaging SPR is based on Au film-based DNA on SPR substrates.
Used in some embodiments to monitor the binding of analytes to microarrays such as probe microarrays. In a normal scanning SPR, S
The SPR reflectivity is measured by changing the incident angle within the angular range where PR occurs. Therefore, one measurement of the total SPR substrate SPR reflectivity is obtained at each angle of incidence. In contrast, in the imaging SPR embodiment, a fixed angle of incidence is used and light undergoing total internal reflection is imaged, preferably on the surface of a charge coupled device (CCD). The image obtained spatially depicts the SPR reflectivity change. Changes in SPR reflectivity result from localized changes in refractive index near the surface of the SPR substrate; these latter changes, in turn, result from adsorption or desorption of substances at the surface of the SPR substrate. Therefore,
The reflectance image reveals the sites on the SPR substrate where such adsorption or desorption occurred.

In a preferred embodiment, the colloidal Au reagent described above is used as a contrast agent for imaging SPR. The image is preferably captured by binding the colloidal Au-tagged reagent to the Au film at the angle at which the greatest increase in reflectivity is observed. In the resulting image, the areas of highest intensity represent the areas of the Au film that received the greatest increase in reflectivity, and therefore the highest amount of colloidal Au bound. As mentioned above, the angle of maximum reflectivity change (maximum increase and decrease in reflectivity) was determined from the SPR profile obtained when colloidal Au was bound to the Au film (ie, the "naked" Au film (ie, bound). It can be easily determined by subtracting the SPR profile (without the colloid).

In a preferred embodiment, a CCD camera is placed so that the “bare” Au film appears black in the reflectance image. In this way, the change in reflectance is
Higher than background "reflectivity and easily observable.

In a preferred embodiment, the SPR substrate is imaged at an angle of incidence where the reflectance observed by colloidal Au adsorption maximizes. However, colloidal Au
It will be clear that one can also image at the angle of incidence where the reflectance observed by adsorption is maximally reduced .

Since the imaging SPR simultaneously provides reflectance data for multiple discrete Au film areas, this technique allows for SPR-based microarray assays to be performed. In these embodiments, the reagents-such as antibodies, DNA probes, etc.-are localized to specific discrete regions of the Au film. In a preferred embodiment, different reagents are immobilized in each separate area. Methods for arraying biomolecules in this manner on a solid support are well known in the art. The liquid suspected of containing the target of interest is then contacted with an Au film, as described above, as well as a colloidal Au pairing reagent. The Au film is then imaged using an imaging SPR device. The normalized reflectance intensity in each region on the Au film reveals the amount of target bound to it; the location of the reflectance signal reveals the identity of the target. In this way, it is possible to quantify multiple targets simultaneously. Furthermore, it is also possible to obtain kinetic data for multiple targets (eg DNA polymorphisms) simultaneously using the method of the invention.

In some embodiments of the invention, the assay comprises one or more size colloidal A
It is carried out using a mixture of u nanoparticles. In such embodiments, it is necessary to image the SPR substrate at one or more angles of incidence, since the angle of maximum reflectance change varies with nanoparticle size. For example, if using a 50 nm thick Au film, 10 nm colloidal Au becomes localized in the Au film,
A large reflectance change is observed at 70 °, but when the 35 nm Au colloid becomes localized in the film, no change is observed at this angle. However, if the incident angle was raised to 73 °, a large reflectance increase was observed with 35 nm Au-colloid, but not with 10 nm Au-colloid. Therefore, measurements at both angles are used to determine the amount of each particle type present.

In a preferred embodiment, the imaging SPR device is used such that the SPR substrate is horizontally oriented. This is possible by placing the prism bottom upright and facing the horizontal. The SPR substrate can be placed on top of the bottom of the prism and is optically coupled to the prism using, for example, a drop of immersion oil. In a particularly preferred embodiment, the prism is mounted on a turntable to accommodate the adjustment of the angle of incidence.

The horizontal prism geometry provided by the present invention allows for two significant improvements to SPR devices in the field. First, the positioning of the SPR substrate parallel to the laser stage (ie, horizontal, face-up orientation) allows for rapid and parallel introduction of solutions and reagents onto the sample surface. In fact, a very simple experiment is performed with all the solution chemistry contained in the sample droplet. Liquid reagents are also introduced robotically in this geometry, as opposed to the vertical sample positioning of the prior art where the solution had to be cast into the substrate. Second, there is no complicated flow cell for the introduction of liquid, only the sample is placed on top of the prism to capture the image. In this arrangement, the substrate can be quickly interrogated in a continuous fashion,
The same way 6 wells are introduced into a plate reader. Therefore, the possibility of robotic introduction of the substrate is much higher in this horizontal arrangement than in the vertical sample orientation of the prior art.

FIG. 14 illustrates a front perspective view of the preferred embodiment of the imaging SPR arrangement outlined above. The device is mounted on an optical bench 13. Excitation source 1 is preferably a laser, more preferably a 1 mW HeNe laser (632.8 nm, 500: 1 polarization).
Alternatively, it is an Ar ⁺ / Kr ⁺ laser capable of multi-wavelength excitation. The laser light passes through the neutral density filter 2 and then through the linear polarizer 3 to further increase the polarization absorbance. The polarization is parallel with respect to the horizontal plane and therefore the horizontally oriented prisms /
The sample assembly 8 is p-polarized. The two mirrors 4 and 5 direct the light rays upwards (preferably at an angle of 20 °) from the surface of the optical table 13 into the spatial filter 6. The spatial filter 6 spreads the rays, at which point the centers of the rays are made parallel by a plano-convex lens 7. Spatial filter 6 preferably consists of a 60X microscope objective with small holes of 5 μm and preferably spreads the light beam to a diameter of ˜4.5 inches. The plano-convex lens 7 preferably collimates the expanded light rays to a diameter of 3 inches. It is important to note that this dramatic ray broadening makes it possible to illuminate the sample with a wave front that has very little spatial intensity fluctuations. This avoids potential background-subject ambiguity in the acquired image.

The collimated light rays leave the lens 7 and enter the prism / sample assembly 8. 20 °
The upward ray angle provides a 70 ° angle of incidence at the prism / sample assembly 8, which is optimal for high contrast SPR imaging of thin Au films. In the preferred embodiment, the prism / sample assembly consists of a 70 ° -70 ° -40 ° prism whose bottom surface is positioned parallel and away from the optical bench 13. The SPR substrate, which preferably consists of a 50-nm-thick Au film deposited on a microscope slide, is optically matched to the prism bottom surface with a droplet of immersion oil. Coarse positioning of the sample holder was achieved by sliding the prism / sample assembly 8 on a vertically mounted rail, while fine pressure adjustment was made on a micrometer actuated carriage. Attenuation of the expanded light beam Total internal reflection occurs, causing a downward passage of the light beam to the sampling portion of the device. At this non-sharp imaging angle, the reflected ray is compressed almost in one dimension as shown in FIG. This is corrected by remagnification and collimation of the image by a pair of semi-cylindrical plano-convex lenses 10 and 11. The beam then passes through a charge coupled device (CCD) camera 12
It is refocused and collected.

Since the light rays are deflected upwards from the surface optics table 13 by the mirrors 4 and 5, the optical elements 5 to 12 of FIG. 14 preferably provide a robust structure and thus a high stability rail system. Can be placed on top. In the preferred embodiment, mirrors 4 and 5 and the rail system are adjustable to change the incidence and collection angles over the entire useful SPR range (ie, about 45 ° to about 80 °).

Imaging SPR devices are generally considered to be less sensitive than scanning SPR devices. However, using the imaging SPR device of the present invention and the Au-colloid (12 nm) enhancement method, with a dynamic range greater than 5 orders of magnitude,
Allows reliable detection of approximately 8 × 10 ⁷ oligonucleotides / cm ^{2 in} a multi-spot array. This result was obtained without optimizing the hybridization efficiency and shows a 2 to 4 orders of magnitude improvement in sensitivity when compared to the values provided in the literature for scanning SPR devices without Au-colloid promotion. ing. The non-optimized sensitivity of microarray scanning SPR with Au colloid enhancement is at least comparable to that of conventional fluorophore-based methods for target DNA sequence detection in microarrays. However, use of the devices, methods and reagents of the invention will provide greater sensitivity than fluorophore-based systems. Optimization of Au colloid selection (eg size, shape, composition etc.) and hybridization protocol (eg probe, annealing conditions etc.)
It will be the normal work of one of ordinary skill in the art.

There have been significant improvements in geometry and methods for creating surface plasmons. Aspects of the invention directed to the practice of some of these aspects are described below.

Simple SPR interferometers have been described in the literature. For example, Kabashin an
In the work of d Nikitin, the light rays are distributed prior to Kretchsmann-type configuration surface plasmon excitation and recombined with the reflected light. Kabashi
n and Nikitin. Opt. Comm. 150: 5-8 (1998)
. The resulting interference pattern is imaged using a CCD. At the resonance angle of 43.7 °, the minimum change in refractive index that could be detected was Δn = 4 × 10 ⁻⁸ , which was two orders of magnitude lower than what could be detected by measuring the angle shift. In one embodiment of the invention, the measurements could be performed using 12-nm diameter colloidal Au amplification, although Au coverage was too low to be detected by angular shift (apparently based on AFM data. It exists) and can be detected interferometrically. No detectable scattering is expected at such low coverage of small nanoparticles.

In another aspect of the invention, the sensitivity of colloidal Au-amplified SPR is improved by the use of "poor photonic crystals". Photonic crystals are materials that are completely opaque at some wavelengths and completely transparent at others. Surface plasmons have been shown to promote optical transmission through subwavelength hole arrays in thin Ag films. Two anomalous effects have been observed: First, there are distinct transmission minima and maxima, as expected for photon band-gap materials. Second, the transmission through the holes is 1000 times more than predicted by standard hole theory. This indicates that the light flow in the holes is promoted by the coupling with the surface plasmons. The present invention utilizes facilitated coupling between holes and metal nanoparticles within the holes. To do that, a thin Ag film on glass with an array of holes (100 nm diameter) is prepared as described in the literature. The Ag surface is then coated with an organic thiol SAM to allow the glass under the holes to be selectively derivatized with the capture antibody. A sandwich immunoassay is then performed with the secondary antibody tagged with colloidal Au particles. Colloidal Au
The introduction of particles led to large changes in the optical properties of the array of holes (eg reflectivity, transmittance). Greater changes are observed with non-electrical growth of the particles, which almost fills the holes. Since the film is coated with the organic thiol SAM, the metal will only grow in particulate form.

In another aspect of the invention, simultaneous measurement of colloidal Au-SPR scattering over 2π and reflectance as a function of angle is possible. The CCD is mounted on a turntable and can move with the hemispherical prism to measure scattered light at all angles. As a result, there is a total scatter image for each reflectivity data point. Three data characteristics are important. First, because the photodiode collects both scattered and reflected light, a measurement of the scattered intensity reaching the CCD at a given angle of incidence will distinguish the scattered and reflected light. To be able to do. This is an essential pre-requisite for understanding SPR reflectivity at surfaces with immobilized large particles, which exhibit both scattered and reflected components. Second, the scatter image itself is used to determine the size of the colloidal Au nanoparticles bound to the surface as well as to quantify the number of bound particles. Third, there is an angular dependence and spatial distribution in scattering. Both the separation size and the laser excitation angle have a strong influence on the scattering intensity. Wet-Chemical Synthesis of Au Films There is a significant barrier to the requirement for a specific thickness of evaporated Au for high throughput SPR substrate fabrication. To address this problem, the present invention uses a thin A
A rapid wet-chemical method for obtaining u-film is provided. This method is "seeding"
Use the mechanism. In a preferred embodiment, preformed colloidal Au
On the self-assembled monolayer of particles, the selective reduction of Au ³⁺ ions from aqueous solution produces a seeded film. A detailed protocol for wet chemical "seeding" of thin Au films is provided in Example 4. PDMS Microwell Assay In order to rapidly process multiple chemistries on the same surface, there is a need for a method that physically separates each unique chemical interaction and allows each question to be individually addressed. One aspect of the invention relates to the fabrication and implementation of polymeric, evenly distributed microwell arrays. The array of wells is biocompatible, highly flexible, has very low chemical reactivity, can be bonded to any substrate without adhesives, is inexpensive to manufacture and is a readily available material. is there. These properties make the array a very versatile tool in performing and analyzing arrayed chemistries, enabling the simultaneous analysis of multiple surface chemistries by surface plasmon resonance (SPR). Such microwell arrays are also useful for analysis by ultraviolet-visible spectroscopy (UV-vis), surface enhanced Raman scattering (SERS) and other surface sensitive analytical techniques.

Recently, the use of tools in chemistry has increased significantly. The rapidly growing use of combinatorial chemistry and its applications in high throughput screening accounts for the vast majority of array uses in chemistry. Combinatorial chemistry is used in diverse chemical areas such as drug and inorganic compound discovery and new substance synthesis. Arrays are also increasingly used as sensors to perform analyzes of distinct compounds in a chemical mixture, each individual sensor being used to detect a unique compound. Alternatively, individual sensors are combined with others in a sensor array to form a library of components in the sample to be treated. Capillary electrophoresis columns arranged on a microchip are an example of array adaptation to analytical techniques.

Polydimethylsiloxane (PDMS) is a commercially available and inexpensive polymer with desirable properties for microwell arrays. The biocompatibility of PDMS makes it ideal for medical uses such as implants, artificial joints, contact lenses and vascular and skin grafts. Industrial applications include electrocoating, insulators, defoamers, carbon paste electrode media, micromachining, and microfabrication by microcontact printing (pCP) and microfabrication in capillaries (MIMI.
C) is included. The fabrication of the PDMS microwell array of the present invention is centered around 0.
Begin by cutting the sections of an electric breadboard in a square pattern with 046 inch diameter holes spaced 0.092 inch apart. Pour the PDMS prepolymer into a breadboard mold and leave the prepolymer in a plastic petri dish to cure. The PDMS-breadboard composite is removed from the Petri dish and the components are separated by hand. One potential problem with reproducing arrays of this scale is:
Some posts will become separated from the rest of the array. To prevent this, an optical coating of a release agent (eg Dow Corning 230 solution) can be used on the breadboard mold. In addition, a thin layer of polymer is cured on the back side of the mold and can be easily scraped off with a safety razor blade. The PDMS "post array" is then used as a mold. Again, the mold is lowered into a Petri dish (post down) and PDMS is poured up. Place a weight on the mold to ensure proper contact between the top of the post and the surface of the Petri dish. The prepolymer is cured again. Remove PDMS "sandwich" from Petri dish and separate. The new microwell array is then sonicated in soapy water, then methanol, each for 10 minutes. FIG. 17 shows the result of each molding process.

Adhesion tests were performed on glass, doped silicon, polycarbonate and gold deposited on glass. Well arrays were attached to a clean substrate by manually pressing them against the surface and causing electrostatic adhesion. The designated wells were then pipetted with 0.01 M aqueous bromocresol blue solution. Adhesion stability is 10,
Recorded at 20, 60 and 720 minute intervals. Even after 720 minutes, binding to a given surface, no evidence of bromocresol blue leakage was observed with any of the substrates (Figure 18).

Well array masters showed significant integrity and several post masters were used multiple times to replicate well arrays, but did not appear to increase structural defects in appearance. The postmaster was replicated in several different sizes and patterns, but was equally successful and showed no defects. Hole sizes range from 0.001 inch to 0.5 inch, preferably 0.025 inch to 0.1 inch in diameter, with hole densities up to 400 per square inch. Well arrays can be stacked on top of each other and used to form postmasters with longer posts, from which arrays with deeper wells are subsequently manufactured. The dye may be introduced into the elastomer prior to curing, producing a colored well array. The use of a colored array reduces stray and reflected light and is therefore more efficient for analysis when the light source is illuminated through individual wells.

The PDMS well substrate of the present invention serves as an easy fabrication and implementation method to physically separate the individual chemistries on the substrate. Each of these wells can be treated individually and surface chemical interactions can be analyzed without cross-contamination. Therefore, these arrays are readily adaptable to virtually any surface analysis technique. Background Signal Reduction The main source of noise in SPR measurements is the background signal, ie non-specific binding of colloidal Au. One way to further improve the quality of the colloidal reagent is HS (CH ₂ ) ₁₅ CO ₂ H (10%) and HS (CH ₂ ) ₁₀ CH ₂ OH (90%
), The same composition as the thiol used to coat the substrate), coating the Au nanoparticles with a SAM. Since the carboxylic acid on the particles will be extruded by many carbons, it can be easily activated by EDC, but the large weight of alcohol on the surface will eliminate non-specific binding. Also,
It is possible to control the number of biomolecule binding sites available by appropriate dilution of the carboxylic acid. In addition, the biomolecule: particle probe is lyophilized to avoid the possibility of varying the amount of biomolecule: colloidal Au vs. conjugate over time.

Other means of reducing non-specific binding, especially in DNA hybridization experiments, are: controlling temperature, adding non-complementary sequences, adjusting salt concentration, and DNA during hybridization to surface oligos: Reducing the solution concentration of Au vs. conjugate is included. To further reduce non-specificity, longer linkers between the DNA and the surface may be used both on the colloidal Au particles and on the Au film. In addition, the polyethylene glycol moiety can be covalently attached to the carboxy-terminated SAM. Increased sensitivity with secondary amplification Increasing the degree of Au nanoparticle-mediated changes in reflectivity would be achieved by direct amplification of the signal. This is done by increasing the size of the colloidal Au nanoparticles that are brought into the vicinity of the SPR film via the binding reaction. Using both Au ³⁺ and Ag ⁺ (and a suitable reducing agent),
Several different methods of growing immobilized colloidal Au nanoparticles have been shown.

Another method of direct amplification is to use a particle binding cascade, where the binding of a single particle undergoes specific nucleic acid (eg, DNA) interactions, followed by subsequent particle binding. Triggers. In one example, colloidal Au "detection particles" (
DP) is coated with both a monoclonal antibody that recognizes the antigen of interest and a 30-base single-stranded DNA oligonucleotide (A). A first amplification particle consisting of gold colloid coated with two single-stranded oligonucleotides (A 'and B') (
AP-1) was also prepared (eg, by adding a mixture of streptavidin: Au to biotinylated oligo), where A'is wholly complementary to the oligonucleotide on the detection particle, while B'is It is totally non-complementary. Binding of the antigen to the capture antibody leads to DP binding. Subsequent introduction of AP-1 into the solution resulted in multiple AP-1
This results in the binding of particles to DP. A single-stranded oligonucleotide ((
A second amplification particle (AP-2) coated with B) is then introduced, which results in the binding of multi-AP-2 particles to AP-1 particles. In fact, this cascade scheme is expanded to include additional particles.

Reflectivity and Scatter vs. It is important to note that the non-linear response of particle size means that the amplification is also non-linear: three particles that are very closely coupled are optically single large Behaving like a particle would result in a much larger change than that observed with the binding of three small isolated particles. Furthermore, it is possible to induce a network of particles to bind to a single bound particle, but such a network cannot induce network formation by DP itself, thus prior to binding to DP. Network must inevitably exist. Since it is essential that there is no non-specific binding of DP, AP-1 or AP-2, the method by which the cascade of nanoparticles is constructed sequentially provides a method for eliminating non-specific binding of aggregates. It is suitable as a subject.

Alternative cascade schemes may be used that do not require continuous addition of amplifying particles. This is accomplished by designing the oligonucleotides that are attached to the amplification particles so that they are not complementary to each other, but to another single-stranded segment of nucleic acid (eg, DNA). This complementary oligonucleotide can be introduced into solution to activate the cascade resulting in binding of the amplified particles. For example, AP-1 is coated with single-stranded oligonucleotides B and C that are not complementary to each other or to A (coated on DP). As mentioned above, binding of antigen to the capture antibody will result in binding of DP. However, AP-1 present in solution will not bind to DP until the single stranded oligonucleotide A'B '(complementary to oligos A and B) has been introduced. Therefore, AP-2 (also present in solution), a single-stranded oligonucleotide C'D '(complementary to oligos C and D), coated with single-stranded oligonucleotide D, was introduced. It will not bind to DP until. As above, this cascade scheme is expanded to include the addition of additional amplification particles. The advantage of this alternative scheme is that the amplification particles do not have to be added continuously and may be present in solution from the beginning. High-Throughput Screening of Combinatorial Libraries In one embodiment of the invention, particle-stimulated SPR is used in a high-throughput assay for libraries of small molecules. The paradigm of combinatorial chemistry for drug delivery currently creates a situation where the design of functional assays is rate limiting,
If any, very few general applicable solutions are offered.
The simplicity of SPR and the ability to use imaging modalities make SPR an excellent choice.

High throughput applications require higher site density due to SPR measurements. PDMS microwell arrays (> 100 "beaker" density per square inch) provide such high density sites. Such small amount of repetitive pipetting is inconvenient (or impossible) to perform by hand and is accomplished by the use of an arrayer robot. The method uses the robot's eight dispensing needles to place a small spot of HS (CH ₂ ) ₁₁ CO ₂ H. This molecule forms a SAM at discrete locations on the substrate (ie where the dispensing needle comes very close). The rest of the substrate is HS (CH ₂ ) ₁₅
Fill with CH ₃ to create a hydrophobic environment around each (hydrophilic) spot. When the arrayer robot dispenses an aqueous solution, it forms droplets on the hydrophilic patch, which provides the necessary boundaries to work at high site density. Another solution is to use robotic dispensing needles to shape them (but the tip is large), which can be filled with solvent. Example 1 Preparation of evaporated Au films and Au colloids The following materials and methods were used to obtain the data shown in Figures 1-5.

Au colloids with a diameter of <12 nm are described by Grabar et al. J. Ana
l. Chem. 67: 735-743 (1995) and Grabar et.
al. J. Am. Chem. Soc. 118: 1148-1153 (1996)
) (Each of which is incorporated herein by reference in its entirety), by citrate reduction of HAuCl ₄ .3H ₂ O.

Au colloids with diameters> 12 nm were produced by improving the “seed colloid” technique. 12 nm colloid in 1 mL (long axis 12 nm ± 2 nm and short axis 11 nm ± 1
nm, from 758 particle sizing), with 100 mL, 0.01% HAu
Cl ₄ and 500 mL of 38.8 mM citric acid solution were used as the starting material. The resulting sample was analyzed by Transmission Electron Microscopy (TEM) and the sampled 274 particles showed a long axis of 45 nm ± 5 nm and a short axis of 37 nm ± 4 nm. 100 mL of 0.02% HAuCl ₄ and 1.0 mL of 38.8 mM
A second batch of large colloids produced using a citric acid solution has 424
The particles showed a long axis of 59 nm ± 14 nm and a short axis of 52 nm ± 11 nm. A batch of particles with the following dimensions was produced from a similar protocol: 3
From the size classification of 73 particles, 30 nm ± 3 nm (long axis) × 25 nm ± 2 nm (short axis), and from the size classification of 112 particles, 35 nm ± 3 nm (long axis) × 30 nm ±
3 nm (short axis). These were prepared with 100 mL of 0.01% HAuCl ₄ and 5
00 mL of 38.8 mM citric acid solution was used, 3 mL and 2 mL diameter 1
2 nm of colloidal "seed" was added each. Average particle diameter and standard deviation are N
IH Image: v. TEM Image using 1.62 software
Was determined by particle size analysis.

The thin (47-50 nm) Au film was evacuated by a diffusion pump, Edward
AuShot from a resistively heated molybdenum boat (Kurt. J. Lesker) in an Auto 306 thin film fabrication system (99.99%, Jo
Hnson Mathey). Evaporation substrate is 1 inch x
1 inch x 0.02 inch, 10% (to increase the adsorption of Au on the glass
v / v) Polished SF11 glass (n = 1.78, Schott Gl) exposed to a (3-mercaptopropionyl) -trimethoxysilane / CH ₃ OH solution for 30 minutes.
ass Technologies) pieces. Au was coated at a pressure of 1 × 10 ⁶ mbar, rotating the sample constantly at 0.5 nm / s to ensure a homogeneous coating. After evaporation, the film was annealed at 300 ° C. for 5-10 minutes in a homemade oven under a constant flow of argon to reduce the surface roughness of the deposited layer.

The annealed Au film was modified with a 10 mM ethanol solution of alkanethiol (2-mercaptoethylamine (MEA) or 3-mercaptopropionic acid (MPA)) for 30 minutes. Longer soak times did not noticeably improve the efficiency or density of subsequent derivatization steps. Amine modification of colloidal Au (MEA
Immobilization to the (coated) surface was carried out at room temperature with an undiluted aqueous solution. The amine modified surface induced strong adsorption of colloidal Au particles due to the availability of unpaired electrons in the amine nitrogen, as well as ionic interactions with negatively charged particles.

SPR data was obtained using a scanning SPR instrument. Excitation of surface plasmons was achieved using a Kretchsmann-type configuration, where a 1-inch diameter hemispherical prism (SF11 glass, Esco Products) was used with an SF11 substrate on which Au was deposited as previously described and a refractive index. Matched (Cargil
le soaking oil n = 1.78). This assembly was then added to a home-made flow cell (volume -100 mL) where the Au film was exposed to the solution. The SPR excitation source was a cylindrical 5 mW, 500: 1 polarized absorbance HeNe laser (632.8 nm, Melle).
s Griot), which is a 500: 1 visible-optimized linear polarizer (Newp
ort 10LP-VIS). Optical chopper (Sta
nforde Research Systems) is used to modulate the optical signal at a frequency of 2 kHz, which in turn is a lock-in amplifier (Stanford).
Correlation with detection via Research Systems, SR 530).
The rays were focused by a 100-mm focal length (fl) plano-convex (PCX) lens and re-collimated by a 25-mm fl PCX lens, which reduced the ray size to a diameter of 0.4 mm ± 0.1 mm. It was The hemispherical lens is then used to focus the beam of light so that it is re-collimated by the hemispherical prism-sample assembly. The reflected beam passes through the diaphragm and is focused on a silicon photodiode detector (Thor Labs), the signal from which is measured with a lock-in amplifier, which is in phase with the excitation source. Two high resolution (0.001 °) servo motors-driven rotary table (Newport, RT)
Achieved by a home-made θ-2θ platform consisting of M80CC and RTM160CC annular rotary platform, which are mounted together such that the axes of rotation are collinear. The prism / sample / flow cell assembly is then mounted on the θ-2θ stage such that the center of the Au / glass sample is the axis of rotation. LabVIE for platform rotation and data collection
Controlled through a computer interface developed using the W programming language (Version 4.01, National Instrument)
nts). Typical SPR scan is 0.1 or 0.01 ° resolution, 0.5 °
s-1 unit rotation speed, and lock-in time constant of 0.3 s.

Particle densities were determined using a tapping mode atomic force microscope (TM-AFM) and the images were operated in Digital Instruments Nano with tapping mode with an acquisition frequency of 1.5 Hz and a line density of 512.
Obtained using seope IIIA. A standard 200 nm etched silicon probe was used for coverage determination purposes. At least 4 images, 2 for each sample
Obtained from three different areas. To confirm that the surface coverage is uniform, 2
Two 5 μm x 5 μm images were taken ~ 0.5 mm apart. At each position, a 1 μm × 1 μm scan was taken to obtain a higher resolution image. Particle counting was done manually from the captured images. Numbers reported are 2-3
It is the average coverage value obtained from the image.

In all experiments, care was taken to ensure that we were looking at the same region of a particular sample with both SPR and TM-AFT. Therefore, during the SPR experiment, the box was scratched around the irradiated area. The surface coverage in that region was then measured by AFM, which reduced errors due to unequal surface induction over large scales. It is important to note that solution agitation is an important step in obtaining particle-diameter-independent coverage values. Short exposure time (<
1 hour) and the same solution particle concentration, almost equal particle surface concentrations are obtained independent of colloid diameter. This removes the diffusion dependence of surface attachment, leaving attachment probability (which is only slightly affected by particle diameter) as a determinant in the resulting particle number density. Example 2 Sandwich Immunoassay The following materials and methods were used to obtain the data shown in Figures 6-10.

Au films and Au colloids were prepared as described above. Human IgG (h
-IgG), γ-chain specific monoclonal goat anti-human IgG antibody [ah-Ig
G (γ)] and F _c -specific monoclonal goat anti-human immunoglobulin G antibody [
a-h-IgG (F _c )] was obtained from Sigma Immunochemicals.

Protein-Au pair conjugates were synthesized by adding ah-IgG (F _c ) to 5 mL of pH-adjusted colloid, followed by 1 hour incubation on ice with gentle agitation. The pair conjugate was divided into 1 mL fractions in a 1.5 mL microcentrifuge tube and centrifuged at 12,500 g for 45 minutes (Heraeus picoBiof.
age). Remove the pink supernatant from the clear and remove the soft pellet from H2O or 40 mM
Resuspended in phosphate buffer (pH 7.0) at an optical density of ~ 3. Paired conjugates can be stored between 2 and 8 ° C for several days without loss of activity.

The flocculation profile was used to determine the amount of protein required to coat the exterior of the Au particles, and thereby inhibit colloid aggregation, ah
It was constructed for -IgG (F _c) -Au colloidal versus conjugates. The solution is ah
-A portion (0-72 μg) of a 0.75 mg / mL stock solution of IgG (F _c ) was added to 6 μ
Adjusted to pH 8.0 using 0.1M NaOH in increments of g (EM
Science Color-pHast Indicating Plate, pH 2-9), 1.0 mL of 10
mm diameter colloid solution. The sample was adjusted to volume 1.150 mL with deionized H ₂ O and 150 μL of 1.0 M NaCl was added to each. The solution was stirred and the optical spectrum was recorded after 10 minutes. For 10 mm colloid, 31.5 μg ah-IgG (F _c ) / mL colloid was determined to inhibit aggregation.

[0103]   To immobilize ah-IgG (γ) (and other proteins) on Au film
In order to introduce a carboxylic acid group on the Au surface, the Au surface is treated with MPA as described above.
Qualified The formation of the active ester on the Au surface is 100 μL of 100 mM, p
H5.5, (1-ethyl-3- (3- (dimethylamino) propyl) carbodi
Reacting an imide hydrochloride (EDC) solution with a carboxylated Au surface for 15 minutes
Achieved by To stabilize the reactive surface by substituting the imide moiety, 5
0 μL of sulfo-N-hydroxysuccinimide (S-NHS) (40 mM, p
H7) is subsequently injected into the flow cell. S-NHS and 15 minutes incubation
After rinsing, the cell is rinsed with 10 mL of buffer solution (85 mM, phosphoric acid, pH 7) and fixed.
Inject a 1.0 mg / mL solution of the protein to be solubilized. The surface is a protein solution and 3
Incubate for 0 minutes and wash with another 10 mL phosphate buffer. Not on the surface
The reaction site was blocked by reacting with a 10 mg / mL solution of bovine serum albumin.
Lock it. After further washing with buffer, immunochemical reactions are carried out;
Typical incubation times for solution and conjugate solutions range from 5 to 30 minutes
is there. All SPR profiles are likely to exist between protein solutions
It was obtained as described in Example 1 in buffer to remove significant refractive index differences. Example 3 DNA detection   The following materials and methods were used to obtain the data shown in Figure 13.

[0104]   Trisodium citrate dihydrate, poly (ethylene glycol) bis (3-amido)
Nopropyl) terminated (PEG), 16-mercaptohexadecanoic acid (90%) (M
HA), 3-mercapto-propionic acid (MPA), ethylenediaminetetraacetic acid (
EDTA), and NaOH were obtained from Aldrich. HAuCl_Four・ 3
H₂O was obtained from Acros. NaH₂PO_Four, Na₂HPO_Four, Dodecyl sulfate
Sodium (SDS, 95%), NaCl, KCl, concentrated HCl, HNO₃, H₂S
O_FourAnd 30% H₂O₂J. T. Baker Inc. Purchased from. 3-
Mercaptopropylmethyldimethoxysilane (MPMDMS) and 3-amino
Propyl-trimethoxysilane (APTMS) is a united chemical
1 Purchased from Technologies. MgCl₂, Spectrum CH₃ COH and CH₃OCH₃Was obtained from EM Science. CH3CH ₂ OH was purchased from Pharmco. 1-ethyl-3- (3-dimethylamido
Nopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide
(NHS) and streptavidin were obtained from Pierce. Restriction enzyme H
infl was purchased from Biolabs. HPLC purified oligonucleotide
Integrated DNA Technology or Pensylva
nia State University Nucleic Acid Fa
Purchased from Cility (Chart I). All reagents should be further purified.
It was used as received. H used₂O is distilled, followed by B
Purified using the arnstead Nanopure system. Hybrid
The lysis buffer was 10 mM phosphoric acid (pH 7.0) containing 0.3 M NaCl.
) Prepared as a buffer. 10 μM non-complementary 12-mer oligonucleotide
Was added as a blocking reagent. 25 mM Tris-HCl (pH 7.8)
, 100 mM KCl, 10 mM MgCl₂Are mixed and used as enzyme buffer.
Was used. Au (99.99%) shot and Cr wire used for evaporation
Is Johnson-Matthey Corp. Obtained from SF11 glass
Slides (Schott Glass Technologies, n = 1.
78) was used for SPR scanning experiments and was performed using Fisher Precleaned Microscopy.
Mirror slides (BK: 7, n = 1.515) were used for imaging experiments.

DNA: Au pair conjugate production. 12-nm diameter colloidal Au was prepared by citrate reduction of HAuCl ₄ · 3H ₂ O as described in Example 1. The average particle diameter was determined by transmission electron microscopy (TEM), which showed a standard deviation of less than 10%. The optical spectrum of the colloidal Au solution was recorded using a HP8453 spectrophotometer.

Thiol-labeled DNA: Au pair conjugates are described by Mirkin et al. , Natu
re 382: 607-609 (1996), Elghhanian et al.
． , Science 277: 1078-1081 (1997), Storho.
ff et al. ． J, Am. Chem. Soc. 120: 1959-196
4 (1998), Storhoff & Mirkin. Chem, Rev. 9
9: 1849-1862 (1999) and Mucic et al., J. Am. A
m. Chem. Soc. 120: 12674-12675 (1998), each of which is incorporated herein by reference in its entirety. 36 μM 12-mer (100 μM) oligonucleotide probe S3 was added to 1 ml of Au colloid solution. After standing for 16 hours, the solution was left for 2 days in a 0.1 M NaCl solution (pH 7, 10 mM phosphate buffer). The DNA: Au pair conjugate was removed by centrifugation (4
5 minutes, 12,500 xg) was washed. The supernatant is discarded, then the red pellet is reconstituted to 0.
It was washed with 1M NaCl solution. After the second centrifugation, the pellet is 0.3M N
Reconstituted at the original concentration in the aCl / phosphate hybridization solution. Paired conjugates are used fresh when prepared and optical spectra are always recorded to ensure that the solution concentrations are consistent.

Biotinylated DNA: Au pair conjugates can be prepared from streptavidin: Au pair conjugates. A solution of Au particles diluted 1: 1 with H ₂ O and adjusted to pH 8.5 with 0.1 M NaCl was added to a 40 mg 1 mg / ml streptavidin stock solution. After incubation on ice for 1 hour, the solution was centrifuged to separate unbound protein (12,500 xg, 45 minutes). 200 μL of biotinylated oligo probe (10 μM) was incubated with the conjugate for 30 minutes followed by 2
The supernatant was removed by performing a second centrifugation and resuspended in a hybridization buffer. Again, optical spectra were taken to ensure agreement of solution concentrations, as was done with the thiol-labeled DNA pair conjugate.

Successful incorporation of the oligonucleotide probe onto the surface of the Au colloid was confirmed by M
irkin et al. , Nature 382: 607-609 (1996).
), Elghian et al. , Science 277: 1078-
1081 (1997), Storhoff et al. ． J, Am. Chem
． Soc. 120: 1959-1964 (1998), and Storhoff.
& Mirkin. Chem, Rev. 99: 1849-1862 (1999
), Each of which is hereby incorporated by reference in its entirety. Briefly, flocculation was induced by mixing the S3: Au pair conjugate solution with a ligating oligonucleotide capable of hybridizing to two different copies of S3, and then monitoring the ligating oligonucleotide. The optical spectrum of the mixture showed the characteristic absorption of Au colloid at 524 nm, but the addition of the linking oligonucleotide led to a red shift of λ _max with a marked decrease in intensity. This is due to the formation of nanoparticle aggregates. DNA
By gradually raising the solution temperature above the melting point of the duplex, dehybridization occurs, causing dissociation of the colloidal Au network. As a result, an increase in optical intensity at 260 nm (the characteristic absorption of free oligonucleotide in solution) was observed. DNA: Au pair binders that do not exhibit reversible flocculation were discarded in this test.

The surface is manufactured as detailed in Example 1. 10 mM MHA / C ₂ H ₅ OH
A self-assembled monolayer was applied to the Au surface by immersion in the solution overnight. Amine-labeled 12-mer oligonucleotide (having a sequence complementary to half of target S2 (
S1) was attached to the carboxylic acid groups on the surface via EDC / NHS crosslinking. next,
Amine-labeled PEG (1%) was introduced to block unreacted sites, followed by washing and target analyte (24-mer DNA (S2
)) Was incubated for 2 hours. In unamplified experiments, an untagged DNA probe (S3) with a complementary sequence to the other half of S2 was added and incubated with the surface. In amplification experiments, SPR surfaces were loaded with Au particle-tagged S3 probe and incubated with the surface. In both cases, hybridization was carried out for 1 hour, 0.2% SDS / 0.1M
Rinse well with NaCl.

Scanning SPR data was collected using the apparatus as described in Example 1. Mini Perista Pump (Intech Laboratories.In
c. ) Was used to deliver a sample of the analyte solution at 0.1 ml / min to the sample cell. A 5 minute wash at 1 ml / min was always performed before collecting SPR measurements. DNA melting, where the temperature of the sample cell was raised by running warm dehybridization buffer (80 ° C as measured in a water bath) for 20 minutes,
All experiments were completed at room temperature. Due to the thermal gradient in the liquid delivery device, a higher temperature was applied here, 27 ° C. above the melting point of the surface bound DNA duplex. The actual temperature at the surface was estimated to be between 60 and 70 ° C. The sample cell was cooled to room temperature before SPR measurement. Kinetic studies were performed by monitoring SPR reflectivity changes as a function of incubation time at a fixed angle (53.2 °).

Imaging SPR was performed using the apparatus described previously. The data is SP
Plotted as a spatial intensity map of the R substrate surface, where increasing intensity indicates increasing SPR response. The integrated intensity from each sample cell was calculated from the spatial intensity map, the baseline intensity was subtracted, and each signal was normalized to the area of the sample cell.

UV-Vis Absorption Spectroscopy: UV-Vis spectra were collected using a HP8453 Diode Array Spectrophotometer equipped with a HP89090A Peltier temperature controller. By changing the solution temperature at 25-65 ° C (1 ° C / step), DN
The A melting assay was tested by monitoring the change in optical intensity at 260 nm. Hybridization buffer was used as a control. Example 4 Wet-Chemical Synthetic Glass Cover Glasses (n = 1.515, Fisher) of thin Au film in a'piranha 'bath (3
0% H ₂ O ₂ is mixed with concentrated sulfuric acid H ₂ SO _{4 in} a ratio of 1: 4. Care must be taken when handling this solution due to its highly oxidizing nature). (3-Mercaptopropyl) methyldimethoxysilane (MPMDM, United Chemical Technology) self-assembled by exposing clean glass slides to an ethanol solution of 1-10 mM silane.
es) for functionalization. The functionalized slides were placed in a 17 nM solution of 12 mm diameter colloidal Au until a surface coverage was obtained giving an absorbance of 0.1 AU at 520 nm. The colloidal Au-modified surface was then treated with 500 mL of 0.5 mM N 2
H ₂ OH. Place in HCl (Aldrich) aqueous solution, orbital shaker, 150
100 mL of 0.06% HAuCl ₄ (Acros) with stirring at revs / min.
) Was added. Stirring is continued for 20 minutes, with a smooth, reflective Au on the monolayer.
The surface was created. Example 5 Synthetic film thickness of PDMS microwell array is as described in Example 1, using a standard 200 nm etched silicon probe, an acquisition frequency of 1.5 Hz and a line density of 512, tapping mode atomic force microscope (TM). -AFM) was determined by measuring the step height at the engraved spot on the surface.

PDMS (Dow Corning Sylgard 184, 10: 1 elastomer to curing agent ratio) was mixed, degassed using a vacuum dessicator, and poured onto a 1 inch square electric breadboard master and a small petri dish. Placed in a dish, the composite was degassed using a vacuum desiccator and cured overnight in an oven at 50 ° C. After cooling, remove the new PDMS "master" from the electric breadboard. This new "master" was used to form the final template for PDMS. Additional PDMS was poured between the new master and petri dishes, degassed and cured overnight in the oven. To ensure constant contact with the bottom of the Petri dish, place the aluminum block on the glass microscope slide on top as a weight. The PDMS sandwich is removed from the oven, cooled, and peeled off to reveal the PDMS replica.

[Brief description of drawings]

FIG. 1 shows the change in SPR profile of a 50 nm thick Au film SPR substrate caused by adsorption of 11 nm diameter colloidal Au nanoparticles.

FIG. 2 shows how adsorption of colloidal Au nanoparticles of size 30 nm-59 nm changes the SPR profile of 47 nm thick Au film.

FIG. 3 shows how the SPR profile of Au films changes with different surface coverage of 35 nm (FIG. 3A) and 45 nm (FIG. 3B) colloidal Au nanoparticles.

FIG. 4 shows the differential SPR profile (incident angle versus percentage change in reflectance (Δ)) for the data shown in FIG.

FIG. 5 plots the maximum change in reflectance as a function of number density for colloidal Au nanoparticles of diameter 30 nm-59 nm.

FIG. 6 schematically shows an example of a sandwich immunosensing assay.

FIG. 7 shows a sandwich immunoassay SPR profile obtained using a secondary antibody (FIG. 7A) and a secondary antibody pair-coupled to colloidal Au nanoparticles.

8 is a differential SPR profile (Δ for the data shown in FIG. 7).
The percent reflectance versus incident angle) is shown.

FIG. 9 shows reflectance time change for immobilization of both un-paired and Au-paired secondary antibodies.

FIG. 10 shows changes in colloidal Au-induced plasmon shift with various concentrations of target analytes in immunosensing.

FIG. 11 shows that the Au film has SiO ₂ of 15 nm (FIG. 11A), 2
11 shows the gradual enhancement of the SPR response that occurs with 12 nm colloidal Au coated on and adsorbed on 4 nm (FIG. 11B) and 33 nm (FIG. 11C) thin layers.

FIG. 12 schematically shows an example of a nucleic acid hybridization assay.

FIG. 13 shows the SPR profile of nucleic acid hybridization assays performed using the scheme of FIG.

FIG. 14 schematically shows an example of an imaging SPR device provided by the present invention.

FIG. 15A shows the SPR min for the following modifications: mixed SAM (90% 11-mercapto-1-undecanol: 10% 16-mercaptohexadecanoic acid / Au: electrostatically immobilized protein. A / anti-IL-5
Complex (a); 0.6 ng / mL IL5 / 25 ng / mL HAS (b); I
1-5: Au (with 0.5% Blott blocker) (c). FIG. 15B shows the SPR minimum in a similar experiment, but 600 ng / mL / 25 mg / m.
LHSA has been introduced.

16 shows different ratios of Na2O3 and HavCly: (a) 1.
Figure 3 shows a UV-Vis spectrum of a colloidal dispersion made by mixing 3: 1, (b) 1.2: 1, (c) 1.4: 1 and (d) 2.0: 1. ing.

FIG. 17 shows the components of a PDMS microwell array fabrication process, from left to right: (A) 1 inch square electric breadboard “master”, (B) PDMS post “master”, and (C) Electric breadboard P
DMS well array replica. The component is placed against a dark background to promote the properties formed from colorless PDMS.

FIG. 18 shows a time course photograph of bromocresol blue in selected wells of a PDMS array; (A) time = 0, (B) time = 3.
0 minutes, (C) time = 60 minutes, and (D) time = 24 hours.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 21/33 G01N 33/543 595 33/543 595 37/00 103 37/00 103 C12N 15/00 F ( 31) Priority claim number 09 / 629,790 (32) Priority date July 31, 2000 (July 31, 2000) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, R O, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Goodrich, Glen State of Pennsylvania, USA 16801, State College ， Shellers Bend 3181 ， Number (72) Inventor He, Lin, USA 94087, California, 94087, Sunnyvale, Granger Terrace 515, Unit 3 (72) Inventor, Rion, El Andrew, Georgia 30066, Marietta, Brusade Way 1675 (72) Inventor Musik, Michael Dee Huntingdon Valley, Pennsylvania, USA Meadowbrook Drive 616 (72) Inventor Hollyway, William Dee, United States Georgia 30308, Atlanta, Myrtle Street 848, Nanver 10 F Term (reference) 2G057 AA02 AB01 AB03 AB04 AB07 AC01 BA01 BA03 BB06 2G059 AA05 BB12 BB13 CC16 EE05 EE12 FF04 GG01 GG04 GG08 HH02 HH03 HH06 JJ01 JJ11 JJ12 JJ19 KK01 KK04 MM02 MM03 4B024 AA11 AA19 CA01 CA09 CA11 HA13 HA14 4B029 AA07 BB20 CC01 CC02 CC03 CC08 FA12 FA15 4B063 QA01 QQ42 QQ52 QR32 QR35 QR38 QR55 QR56 QR8 2 QS34 QS39 QX10

Claims (48)

[Claims] 1. A microwell array comprising a number of spatially separated microvolume wells, wherein the array comprises PDMS. 2. The microwell array of claim 1, comprising between 10 and 200 wells. 3. The microwell array of claim 1, wherein the array is associated with a thin metal film that serves as the bottom surface of the multiple wells. 4. The microwell array of claim 3, wherein the thin metal film is a surface plasmon resonance active metal. 5. The microwell array of claim 4, wherein the thin metal film is selected from the group consisting of Au, Ag, Al and Cu. 6. The microwell array according to claim 3, wherein a glass surface is coated with the thin metal film. 7. A method of attaching a number of reagents onto spatially separated areas of a surface, comprising: providing a microwell array template on the surface, wherein the microwell array consists of PDMS, and Where the array provides a number of spatially separated wells and the bottom of each well is an area of the surface; consisting of introducing a reagent into the wells associated with the surface; and removing the template. How you are. 8. The method of claim 7, wherein the surface is a thin metal film. 9. The method of claim 8 wherein the thin metal film is a surface plasmon resonance active metal. 10. The method of claim 9, wherein the thin metal film is selected from the group consisting of Au, Ag, Al and Cu. 11. The method of claim 8 wherein the thin metal film is coated on a glass surface. 12. A method of performing an assay for an analyte in a test solution that will contain the analyte: providing a surface plasmon resonance active surface; associating a molecule with the surface, wherein the molecule is Is associated with colloidal metal nanoparticles; contacting the surface with the test solution, where the presence or absence of the analyte changes the relative distance between the surface and the nanoparticles; and the SPR profile. A method comprising detecting the distance by measuring. 13. The method of claim 12, wherein the surface plasmon resonance active surface is a thin metal film. 14. The method of claim 13, wherein the thin metal film is selected from the group consisting of Au, Ag, Al and Cu. 15. The method of claim 13 wherein the thin metal film is coated on a glass surface. 16. The colloidal metal nanoparticles are Au, Ag, Al and Cu.
13. The method of claim 12, comprising a metal selected from the group consisting of: 17. The colloidal metal nanoparticles are substantially spherical.
The method described in 2. 18. The method of claim 17, wherein the colloidal metal nanoparticles have a diameter of 1 nm to 150 nm. 19. A method of performing a multiplicity of assays for a multiplicity of said analytes in a test solution that will contain the analytes: providing a surface plasmon resonance active surface; Associated with a surface; contacting the surface with the test solution, wherein the presence or absence of an analyte is associated with a specific type of ligand at or near the surface, a particular type of colloidal metal nanoparticles Occurs; and measuring the SPR profile determines the association between the particular type of ligand and the particular type of colloidal metal nanoparticles, where the SPR profile is between different types of colloidal metal nanoparticles. A method that consists of being distinguishable by. 20. The method of claim 19, wherein the types of colloidal metal nanoparticles are distinguished by their size. 21. The method of claim 19, wherein the types of colloidal metal nanoparticles are distinguished by their composition. 22. The method of claim 19, wherein the surface plasmon resonance active surface is a thin metal film. 23. The method of claim 22, wherein the thin metal film is selected from the group consisting of Au, Ag, Al and Cu. 24. The method of claim 22, wherein the thin metal film is coated on a glass surface. 25. The colloidal metal nanoparticles are Au, Ag, Al and Cu.
20. The method of claim 19, comprising a metal selected from the group consisting of: 26. The colloidal metal nanoparticles are substantially spherical.
9. The method according to 9. 27. The method of claim 26, wherein the colloidal metal nanoparticles are 1 nm to 150 nm in diameter. 28. A method of performing an assay for an analyte in a test solution that will contain the analyte: providing a surface plasmon resonance active surface; performing the assay at or near the surface, The presence or absence of colloidal metal nanoparticles here indicates the presence or absence of the analyte in the test solution; measuring the presence or absence of the nanoparticles by measuring the SPR profile of the surface with an interferometer SPR. A method consisting of detecting. 29. The method of claim 28, wherein the surface plasmon resonance active surface is a thin metal film. 30. The method of claim 29, wherein the thin metal film is selected from the group consisting of Au, Ag, Al and Cu. 31. The method of claim 29, wherein the thin metal film is coated on a glass surface. 32. The colloidal metal nanoparticles are Au, Ag, Al and Cu.
29. The method of claim 28, comprising a metal selected from the group consisting of: 33. The colloidal metal nanoparticles are substantially spherical.
The method according to 8. 34. The method of claim 33, wherein the colloidal metal nanoparticles are 1 nm to 150 nm in diameter. 35. A method of performing an assay for an analyte in a test solution that will contain the analyte: providing a surface plasmon resonance active surface; performing the assay at or near the surface, The presence or absence of colloidal metal nanoparticles here indicates the presence or absence of the analyte in the solution; and where the colloidal metal nanoparticles consist of AuS / Au shell particles; and of the surface A method comprising detecting the presence or absence of the nanoparticles by measuring the SPR profile. 36. The method of claim 35, wherein the surface plasmon resonance active surface is a thin metal film. 37. The method of claim 36, wherein the thin metal film is selected from the group consisting of Au, Ag, Al and Cu. 38. The method of claim 36, wherein the thin metal film is coated on a glass surface. 39. The colloidal metal nanoparticles are Au, Ag, Al and Cu.
36. The method of claim 35, comprising a metal selected from the group consisting of: 40. The colloidal metal nanoparticles are substantially spherical.
The method according to 5. 41. The method of claim 40, wherein the colloidal metal nanoparticles are 1 nm to 150 nm in diameter. 42. A high throughput screening method for combinatorial libraries for identifying compounds having a positive response to a preselected activity: providing a surface plasmon resonance active surface; Of the combinatorial library, whereby multiple components of the combinatorial library are attached to the surface, and where the positive response of the preselected activity is the presence of colloidal metal nanoparticles bearing the surface. Or associated with the absence; and detecting the presence or absence of the nanoparticles by measuring the SPR profile of the surface to identify compounds that have a positive response to the preselected activity. A method made of. 43. The method of claim 42, wherein the surface plasmon resonance active surface is a thin metal film. 44. The method of claim 43, wherein the thin metal film is selected from the group consisting of Au, Ag, Al and Cu. 45. The method of claim 43, wherein the thin metal film is coated on a glass surface. 46. The colloidal metal nanoparticles are Au, Ag, Al and Cu.
43. The method of claim 42, comprising a metal selected from the group consisting of: 47. The colloidal metal nanoparticles are substantially spherical.
The method described in 2. 48. The method of claim 47, wherein the colloidal metal nanoparticles are 1 nm to 150 nm in diameter.