Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

• This invention relates to methods for porous to porous silicon and to method for preparing it for bio-molecular interaction studies.
• An optical biosensor is an optical sensor that incorporates a biological sensing element.
• optical biosensors have become widely used for sensitive molecular binding measurements.
• the targets When using labels to monitor these interactions a fluorescent, colorimetric or some other signal is generated by an additional molecule or moiety that is attached to the target or receptor which gives a signal when the interaction takes place.
• This so called label (or tag) is present only to detect the interaction and is not part of the interaction of interest per se.
• the receptor and target binding are monitored directly using untagged biomolecules.
• a variety of technologies exist in the art to detect binding without labels including surface plasmon resonance (SPR) and white light interferometery using porous silicon.
• SPR surface plasmon resonance
• instrument architectures which can used. These include plate readers and flow cells. In the case of plate readers a well plate (or micro well plate or micro titer plate) is used to house the biochips and fluids which are used for the label free binding studies. This allows for parallel analyses of several types of data. Alternatively flow cells house biochips in, typically, a microfluidic cell which routes fluid over the region of the biochip where the binding interaction takes place.
• SPR surface plasmon resonance
• a resonant mirror system also relies on changes in a penetrating evanescent wave.
• This system is similar to SPR and, like it, binding reactions between receptors and analytes in a region extremely close to the back side of a special mirror (referred to as a resonant mirror) can be analyzed by examining light reflected when a laser beam directed at the mirror is repeatedly swept through an arc of specific angles.
• resonant mirror systems are expensive and impractical for many applications.
• U.S. Patent No. 6,248,539 discloses techniques for making porous silicon and an optical resonance technique that utilizes a very thin porous silicon layer within which binding reactions between ligands and analytes take place.
• the association and disassociation of molecular interactions affects the index of refraction within the thin porous silicon layer.
• Light reflected from the thin film produces interference patterns that can be monitored with a CCD detector array. The extent of binding can be determined from change in the spectral pattern.
• Kinetic binding measurements involve the measurement of rates of association (molecular binding) and disassociation.
• Analyte molecules are introduced to ligand molecules producing binding and disassociation interactions between the analyte molecules and the ligand molecules.
• Association occurs at a characteristic rate [A][BJk 0n that depends on the strength of the binding interaction Ic 0n and the ligand topologies, as well as the concentrations [A] and [B] of the analyte molecules A and ligand molecules B, respectively.
• Binding events are usually followed by a disassociation event, occurring at a characteristic rate [A][B]koff that also depends on the strength of the binding interaction.
• optical biosensors have been used as an alternative to conventional separations-based instrumentation and other methods.
• Most separations-based techniques have typically included 1) liquid chromatography, flow-through techniques involving immobilization of capture molecules on packed beads that allow for the separation of target molecules from a solution and subsequent elution under different chemical or other conditions to enable detection; 2) electrophoresis, a separations technique in which molecules are detected based on their charge-to-mass ratio; and 3) immunoassays, separations based on the immune response of antigens to antibodies.
• separations methods involve a variety of detection techniques, including ultraviolet absorbance, fluorescence and even mass spectrometry.
• the format also lends itself to measure of concentration and for non-quantitative on/off detection assays.
• porous silicon biochips are fabricated in the form of a Fabry-Perot cavity where changes in the white light interference spectrum are used to deduce the time course of the biomolecular interaction.
• This porous silicon biochip is ideally suited for use with the non-invasive, non-destructive, label free white light probe.
• porous silicon surface would be inappropriate for such biochips for three reasons. First, the porous silicon surface is chemically unstable. That is it degrades under the buffer conditions typically used for biomolecular interaction studies. Second, it is difficult to immobilize the variety of receptors the researcher would want to study on the as formed porous silicon surface.
• targets and/or receptors could non-specif ⁇ cally bind to the surface, even if the interaction the research would like to study, are not present.
• This last point, so called non-specific binding (NSB) is of particular concern when designing appropriate surface coverages for biosensor chips.
• NSS non-specific binding
• biomolecular interactions In general what one wants to study in biomolecular interactions is the specific binding of one biomolecular to another molecule (which may or may not be a biomolecule). Any interaction which binds a molecule to the surface generates a signal. That is, the biochip readout instrument cannot generally distinguish between a specific binding event (between target and receptor, which is what the researcher wants to study) and between target and surface. A proper biochip surface coverage must minimize this later interaction.
• the present invention provides a method which renders the 3D surface of the insides of the pores of porous silicon biochip appropriate for conducting studies on biomolecule interactions without labels.
• the method includes a first step in which an as prepared Si-H surface is converted to a silica surface. This silica surface is then silanized in a process that coats the surfaces of the pores with silane.
• the silane coated surfaces are then coated with one of a variety of intermediate moieties for the purpose of minimizing non specific binding and to allow for easy immobilization of receptors.
• a preferred moiety are polymers cut from polyethylene glycol (PEG).
• PEG polyethylene glycol
• the PEG molecules are of a variety of lengths between 4 and 60 monomer units.
• FIG. IA is a general description of the present invention.
• FIGS. IB and 1C are two preferred embodiments of the present invention.
• FIG. 2 shows an example of a silanizing reagent (GOPTS).
• FIG. 3 shows the reaction of GOPTS with the Silica surface.
• FIG. 4 shows how the thickness of the GOPTS layer plateaus after a certain number of
• FIG. 5 shows an example hydrophilic reagent polyethylene glycol (PEG).
• FIG. 6 shows the reaction of hetero bifunctional PEG with the silanized poSi chip
• FIG. 7 shows the concept of variegated PEG length to increase surface density
• FIG. 8 shows the preparation of an benzaldehyde surface using thiol chemistry
• FIG. 9 shows a scheme for single point protein attachment to a surface using ex situ cross linking.
• a preferred embodiment of the present invention is a method for preparing porous silicon chips for use in a biosensor described in parent patent applications Serial No. 11/180,349 filed 7/13/2005, Serial No. 10/631,592 filed 7/30/2003 and Serial No. 10/616,251 filed
• porous silicon biochips are fabricated in the form of a Fabry-Perot cavity where changes in the white light interference spectrum are used to deduce the time course of the biomolecular interaction.
• This porous silicon biochip is ideally suited for use with the non-invasive, non-destructive, label free white light probe.
• porous silicon surface would be inappropriate for such biochips for three reasons. First, the porous silicon surface is chemically unstable. That is it degrades under the buffer conditions typically used for bio-molecular interaction studies. Second, it is difficult to immobilize the variety of receptors the researcher would want to study on the as formed porous silicon surface.
• targets and/or receptors could non-specifically bind to the surface, even if the interaction the research would like to study, are not present.
• This last point, so called non-specific binding is of particular concern when designing appropriate surface coverages for biosensor chips.
• bio-molecular interactions In general what one wants to study in bio-molecular interactions is the specific binding of one bio-molecular to another molecule (which may or may not be a bio-molecule). Any interaction which binds a molecule to the surface generates a signal. That is, the biochip readout instrument cannot generally distinguish between a specific binding event (between target and receptor, which is what the researcher wants to study) and between target and surface. A proper biochip surface coverage must minimize this later interaction.
• Porous silicon contains pores which in the preferred embodiment have an aspect ratio in the range of 30-80 and have pore diameters near 80 nm.
• the solution phase methods taught in for example US Patent 5,436,161 would not successfully coat the insides of these pores.
• the as prepared poSi material is quite hydrophobic and is not amenable to thorough wetting by aqueous solutions.
• the as prepared material is not stable to typical buffering conditions as the thin walls of the material are readily dissolved by solutions near neutral pH.
• material which reacts well with silica tends to also be susceptible to polymerization. This polymerization can easily clog the pores of the material and render it useless for label free biding studies.
• silica is then protected with a silane (FIGlA-B) which generally leaves a hydrophic surface not easily reacted with acqueous reagents. This surface is then rendered hydrophilic (FIG IA-C) through where a number of molecules could be used.
• Porous silicon biochips are formed by anodic etching in HF acid solution as described previously in the parent applications cited in the first paragraph of this specification which are incorporated herein by reference. This etching process leaves a surface where the silicon bonds are terminated with hydrogen as shown at A in FIG. IA.
• This Si-H surface may be converted to a silica surface as shown at B in FIG. 1 (A 5 E & H) by a variety of means including baking in an oxygenated atmosphere at 200° C, O 2 plasma cleaning, or soaking in water (with or without heat). Applicants' preferred technique is the baking process, though other processes are possible.
• the Si-O " surface is then silanized by coating it with a silane which protects the porous silicon surface from degradation as well as allows further reactions to take place.
• the proper method for silanizing the porous silicon surface must account for the high aspect ratio of the pores, which in the preferred embodiment ranges from 30-80. To accomplish this, a multi-step process is preferred which is designed to completely coat the pore surface, while avoiding self polymerization of the silane reagent.
• a tri-alkoxy silane e.g. 3-glycidoxypropyl tri-methoxy silane - GOPTS see FIG. 2
• MMD molecular vapor phase deposition
• an amount of GOPTS is introduced which is not enough to cover the entire surface.
• FIG. 3 The GOPTS molecule has four reactive moieties and care needs to be taken to avod polymerizing the material.
• step C of FIG IA is performed.
• an epoxide group of the GOPTS is used to react one of a variety of 'intermediate' moieties whose purpose is to minimize non-specific binding and allow for easy immobilization of receptors.
• the preferred embodiment makes use of a polymer 'cut' from a polyethylene glycol reaction as indicated in FIG. IB(G).
• PEG molecules of a variety of lengths are used with the mean PEG length (see n in FIG. 5) varied between 4-60 monomers.
• PEGs may be applied in a variety of ways to the silanized surface (see FIG. 6).
• the PEG molecules may be directly placed on the surface and then melted at high temperature (75- 125°C).
• the PEG molecules may be dissolved in an organic solvent such as di-methyl formamide (DMF) and spin cast on the wafer. The solvent is then removed by evaporation and the PEG reacts again with heat (75° C).
• the applicant preferred embodiment is to use heat.
• the PEG 'cuts' (PEG V , or variegated length PEGs) used in this implementation have a distribution of molecular weights. Indeed it is this fact which is crucial to minimizing the non-specific binding on the biosensor chip.
• the process leaves a carboxyl surface which can be used for immobilizing biomolecules through several R groups as shown at D in FIG. IB.
• biomolecules to carboxyl groups
• direct linking with primary amines through succinimide ester of the carboxyl group
• cross linking schemes e.g. hydrazone functionalization of the carboxyl reacting with an aldehyde crosslinker on the biomolecules see e.g. US 6,800,728 which is incorporated herein by reference see FIG. 1C. scheme 2.
• a hydrazone crosslinking scheme a different hetero-bifunctional PEG would be used to react with the epoxide.
• the applicant's preferred embodiment is to use a 24 monomer length PEG which is synthesized in its dimeric form (FIG. 8).
• the disulfide is reduced with a slight excess of Tris(2-carboxyethyl) phosphine (hydrochloride) (TCEP) to give two of the free thiols. These thiols (FIG. 1-J) are reacted as the other hetero-bifunctional PEGs (FIG 1-G).
• the exposed benzaldehyde surface then reacts with molecules containing hydrazines to form a hydrazone bond, though other known reactants to aldehydes may also be used.
• FIG 9A A protein is dissolved in 0.5 mL of water then equilibrated into phosphate buffered saline (PBS) buffer, pH 7.2, using ZEBA columns. 1 equivalent succinimidyl 6- hydrazinonicotinate acetone hydrazone (S-HyNic, SoluLink incorporation San Diego, CA) is dissolved in 0.03 mL anhydrous DMF. After complete solubilization of the S- HyNic reagent, 15 ⁇ L of the solution is added to the dissolved protein, followed by immediate rapid vortexing.
• PBS phosphate buffered saline
• S-HyNic succinimidyl 6- hydrazinonicotinate acetone hydrazone

Applications Claiming Priority (3)

Publications (2)

Family

ID=40549450

Family Applications (1)

Country Status (2)

Families Citing this family (3)

Family Cites Families (4)

• 2008
  • 2008-07-30 EP EP08794883A patent/EP2185480A4/de not_active Withdrawn
  • 2008-07-30 WO PCT/US2008/009214 patent/WO2009048494A1/en active Application Filing

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events