JP2006512564A - Dynamic bio-NEMS sensor and array of bio-NEMS sensors immersed in fluid - Google Patents

Abstract

The bio-NEMS device includes a piezoresistive cantilever having a flexible leg that connects the cantilever to a support and having a biofunctionalized portion at the tip. The bias current applied to the leg is limited by the maximum allowable temperature rise at the biofunctionalized tip. The length of the cantilever is a size selected to minimize background Johnson noise. The catalyzed receptor on the device binds to a ligand with an increased binding rate coefficient. The catalyst is designed by forced evolution to reduce the activation energy of receptor-ligand binding and preferentially bind to the ligand. The carrier signal is injected with a magnetic thin film disposed on a cantilever that is electromagnetically coupled to a carrier signal source. A plurality of NEMS fluid coupled transducers generate a plurality of output signals from which the total output signal is derived, either by averaging or threshoiding. NEMS devices are placed in a microfluidic flow channel and manufactured in a membrane. A binding molecule is attached to the tip of the transducer and a fluff ball is attached to the binding molecule to increase attenuation.

Description

1. TECHNICAL FIELD OF THE INVENTION The present invention relates to the technical field of a pure fluid bioNEMS device and a method for operating the device.

Related Applications This application is a U.S. provisional patent application Nos. 60 / 379,710, 60 / 379,660, 60 / 379,645, 60 / 379,552, 60, filed May 7, 2002. / 379,711, 60 / 379,543, 60 / 379,643, 60 / 379,708 and 60 / 379,681. These applications are hereby incorporated by reference and claim their priority under 35 USC 119.

Incorporation of co-pending applications This application includes US patent application (PAU.34) filed at the same time (invention name: An Apparatus And Method For Vacuum-Based Nanomechanical Energy, Force, And Mass Sensors) and US patent application (PAU.35). ) (Title of Invention: A Method And Apparatus For Providing Signal Analysis Of A Bionems Resonator) is specifically understood to incorporate all of these as described. In addition, the present application is filed with US Patent Application No. 10 / 138,538 (Title of Invention: An Apparatus and Method For Ultrasensitive Nanoelectrochemical Mass Detection) filed on May 3, 2002 and August 9, 2001. US patent application Ser. No. 09 / 927,779 (invention name: Active NEMS Arrays for Biochemical Analyzes) is incorporated by reference in its entirety.

2. Description of Prior Art In recent years, there have been many advances in NEMS and chemical force microscopy (CFM). NEMS research has yielded a group of cantilevers that have a short length and a small thickness that can resonate at high frequencies and high Q values. These NEMS devices exhibit unmatched sensitivity when operated under ideal conditions (low T and reduced pressure conditions). Several groups have used very large instruments (AFM, CFM) to analyze the forces exerted by single molecule interactions ranging from hydrogen bonding and antibody-antigen interactions to covalent bonds. Is conducting research. AFM cantilevers that are modified with biomolecules and interact with modified surfaces or modified magnetic beads exhibit forces on the order of 100 pN for antigen-antibody interactions and about 1-10 nN for covalent bonds Indicates. These groundbreaking experiments show the possibility of measuring chemical phenomena at the probability limit, but also show evidence that it is difficult to realize this possibility with a small, portable and rugged device. Yes.

  What is needed in the present invention is to reduce the size of the cantilever to the size of the NEMS, giving the single molecule sensitivity necessary to build a device with the required temporal response, small volume and single cell performance. Is some way to do. In order to place the NEMS cantilever into the solution at room temperature, it will of course be necessary to modify the detection strategy normally employed for CFM or NEMS. The fluid damps the NEMS cantilever, making resonance unmeasurable and the energy of the solution impacts the cantilever.

  What is needed is to make use of these fluids as a component of this assay for possible obstacles.

  In contrast to conventional CFM, what is needed in the present invention is a method that does not attempt to measure the force of a single (small number) chemical bond by recording the bending of the cantilever.

  What is needed is a type of NEMS cantilever design that uses an integrated sensor to detect the presence of chemical bonds due to the limitations caused by the chemical bond to the large movement of the cantilever that is otherwise driven by heat.

  What is further needed in the present invention is a means of using an array of bio-NEMS cantilevers with systematically different chemical modifications that provide high sensitivity as well as high sensitivity to concentration.

  Furthermore, what is needed in the present invention is the opportunity to assemble and use some method of interpreting the “noise” of rocking as a signal and an in vivo useful and robust assay.

  Recently, not only analysis of gene expression status but also analysis of protein receptors and their ligands have been greatly advanced by the microarray method. For example, thousands of targeted microarrays have become the primary technology used by the drug discovery industry. These microarrays are manufactured by photolithography, microstamping, or microdotting, and a spot array (20-100 □ m) is formed on the substrate. This array is generally read by pouring analytes with fluorescent labels onto the array and then scanning the array with a microfluorimeter to determine the amount of binding. Although these methods are increasingly popular, they are quite unsuitable for applications that require both portability and robustness due to the large size of the reader and the limitations inherent in the fluorescence analysis employed. It has become a way. Furthermore, because they are disposable devices, they cannot be easily adapted to applications that require continuous monitoring. Eventually, these devices rely on significant volumes of analytes, making them unsuitable for the most powerful new advances in drug discovery offered by combinatorial chemistry or the most sensitive assays for gene expression. ing.

  Another goal of the proposed research is to develop a new technology for nanoscale biochips (BioNEMS) that can detect the binding of single biomolecules to their receptors. The literature on growing chemical force microscopy (CFM) modifies AFM to range from single hydrogen bonds and single receptor-ligand interactions to single covalent bonds It shows that the binding force can be measured. Although these force ranges are well within the detection performance of the AFM instrument, AFM cantilevers in solution can reliably track the binding and unbinding of biological ligands and their receptors. It lacks the time response characteristics necessary to become. Perhaps even more importantly, a fairly large device is required to perform AFM / CFM and obtain the well-known sensitivity of AFM to airborne and surface vibrations.

  What is needed is to be as successful as CFM in detecting the interaction force of a single molecule, but to be able to respond quickly enough to scale down to the NEMS scale and track the binding and separation events. It is a kind of technology. Considering the magnitude of chemical force, the most powerful mode in which bio-NEMS operates is the mode that does not directly measure the binding force. What is needed in the present invention is that there is some kind of support that is used in AFM to track with an integral sensor using the ongoing swing of the NEMS cantilever position. Means.

With the present invention, the movement of the NEMS cantilever is used to track the binding and separation events. The basic idea is that a cantilever that does not have a ligand-receptor pair attached to its tip will swing more dramatically than a cantilever that is restricted by a ligand-receptor pair. . Strong ligand-receptor binding can block cantilever movement in part and for a significant amount of time (about t _on for ligand-receptor pairs), changing cantilever movement statistics even with weak interactions Let

  There are a number of advantages to reducing the size of a small NEMS device. The term “NEMS” as used herein means a device having at least one dimension of 1 micron or less than 1 micron. The “NEMS” device may have one or more other dimensions greater than 1 micron. It will also be appreciated that there are often no clear boundaries characterizing devices that are less than 1 micron in size and devices greater than 1 micron. A more important meaning of the term “NEMS” is that the device in question shares sub-micron devices or similar characteristics of operation with similar devices reduced to sub-micron dimensions. As already mentioned, NEMS devices can respond dramatically better to binding and separation kinetics due to their small size. This high frequency response is important in tracking the stochastic nature of receptor-ligand interactions, with most receptor-ligand pairs interacting and binding dramatically, It remains bound (by the exact receptor-ligand pair) for a time in the range of seconds to a few seconds and then separates. A high frequency response (˜MHz) is important when the assay must track biomolecular interactions. The ability of the patch clamp (gigaohm seal) method to analyze the opening and closing of individual membrane channels has dramatically changed our understanding of the physical biochemistry underlying neuronal function, so that only the patch clamp method is the membrane It was possible to decipher the molecular mechanism by recording a large group of channels. We believe that the analysis of biomolecules currently requires a large amount of required substances and even the most sensitive assays are bound by smearing for a specific period and are in exactly the same state . The bio-NEMS is thus balanced by the present invention and advances our biomolecular analysis method to the probability limit accurately.

One of the capabilities of this method is that it uses the thermal motion of the cantilever, which is usually the main constraint of AFM, as the driving force. Furthermore, the noise of cantilever motion decreases as the size of the cantilever decreases. Moreover, because the NEMS device is small in size, an array of detectors ( > 500 cantilevers) can be constructed with a small active volume ( < 100 pL). This latter advantage is very important because it provides the future potential of technology to detect the levels of RNA, proteins and second messengers present in a single cell.

  The bio-NEMS method of the present invention is significantly smaller in size than AFM / CFM and provides the properties necessary for the device to operate. The cantilever motion sensor is integrated with the NEMS cantilever, eliminating the size and density constraints imposed by the optical detection method of cantilever motion used in AFM. As a result, bio-NEMS cantilevers can be much smaller and dramatically more closely packed than those used in AFM.

  As described in the following section on NEMS detection methods, the incorporation of a piezoresistive transducer provides sensitivity much greater than that required to record NEMS cantilever motion in liquid water. As a result, by incorporating it properly, one device with the detector necessary to track the movement of the cantilever, the logic necessary to interpret the movement, and the circuitry necessary to communicate the results obtained. Can be loaded. The current "DNA Chip" or "Proteomics Chip" method, in contrast to its reputation, requires bulky and heavy readers for sensor containers that are several centimeters long and wide to interpret chemical binding. The bio-NEMS method described here promises a package size (approximately DIP dimensions) consistent with the term “chip”, thus providing various applications that are not possible or practical with other methods.

  The purpose of the proposed research is to take advantage of the heat-driven motion of the cantilever and modulate that motion with receptor-ligand interactions. Interpret cantilever motion using knowledge of physics and stochastic biochemistry of NEMS cantilevers in solution. Therefore, in order to construct a practical bio-NEMS of this class, we participate in the fluid mechanics of researchers who are in charge of NEMS assembly, biologists in charge of biochemical modification of the device surface, and NEMS devices. Close cooperation of scientists and information scientists who are good at extracting and analyzing data from arrays is needed.

  The bio-NEMS research effort described here has a number of goals in the fields from basic science to applied science, and aims to develop new nanoscale pure fluid technology. Our goal is to develop, interpret and then improve the technology of bio-NEMS construction and show its new uses.

Examples of applications include:
・ Basic tests on the performance of NEMS in solution.
・ Basic tests on single molecule chemistry.

Cell tests for hormones, growth factors and second messengers. Release of growth factors from cells is generally too low and too small in volume to be analyzed directly by traditional methods.
Use of bio-NEMS as a sensor for the output of combinatorial chemical synthesis of tests for drug discovery.

Use as a highly sensitive “gene chip” or biological hazard sensor for DNA sequence detection.
-Use as a monitor for the concentration of environmental toxins.

The present invention is a piezoresistive cantilever having a length l, a width w, and a tip coupled to the support and extending from the support, the width b having a narrow width and a length. l Defined as a sub-micron bio-NEMS device comprising a piezoresistive cantilever with _one limiting part and a biofunctionalized part at or near the tip. The limiting portion is composed of a plurality of legs having a narrowed width b and coupled to the support. Preferably, two legs are installed and separated from each other at a distance of w-2b.

  The bio-NEMS device further comprises a bias current source applied to the cantilever limiting portion, the magnitude of the bias current being limited by the maximum temperature rise allowable at the biofunctionalization tip.

  The present invention also provides an in-fluid having at least one vibrating cantilever of length l and of a size selected to minimize background Johnson noise associated with the signal intensity emitted by the piezoresistive bio-NEMS device. The present invention relates to improvement of a piezoresistive bio-NEMS device immersed in the water. In one embodiment, the signal strength is based on the level of thermomechanical noise of the piezoresistive bio NEMS device in the fluid. This piezoresistive cantilever has a limiting portion with a width w and a narrowed width b, which is selected to reduce Johnson noise related to the signal intensity emitted by the piezoresistive bio NEMS device Has been.

  The present invention is further characterized by an improvement of a biofunctionalized bio-NEMS device immersed in a fluid, on the bio-NEMS device, a receptor that binds to a target ligand, and its receptor, A catalyst is placed that increases the binding rate coefficient between the receptor and the ligand of interest. The catalyst reduces the activation energy of receptor-ligand binding. In one embodiment, the receptor is designed by forced evolution to bind preferentially to the ligand of interest.

  The invention also includes a carrier signal source, a support, a piezoresistive cantilever coupled to and extending from the support, and the cantilever disposed on the cantilever such that the cantilever is driven by a carrier signal from the signal source and Defined as a submicron device comprising an element that is electromagnetically coupled to the signal source. The element is composed of a magnetic film disposed on a cantilever, and the carrier signal source emits an electromagnetic signal coupled to the magnetic film.

  Furthermore, the present invention processes a plurality of NEMS resonators or NEMS converters (each of which generates an output signal) and a plurality of corresponding output signals from the plurality of NEMS converters to process a collective output signal. signal) is a device with means or circuit. This means averages the plurality of output signals so that the total output signal has an average value. This means checks whether a predetermined fraction of the plurality of output signals exceeds a threshold value within a predetermined time. Since these multiple NEMS transducers are biologically functional, the means can efficiently increase the ligand capture rate by only issuing a comprehensive output signal that indicates an increase in ligand capture rate compared to a single NEMS transducer. To increase.

(式中、k_Bはボルツマン定数であり、Tは流体の温度であり、tは時間でありそしてX₁₂は第一変換器に作用する力F₁に対する第二変換器の変位x₂(t)を示すサスセプティビリティー(susceptibility)である)で定義され、その結果第二変換器のポジションの総合平均(ensemble average)が下記式：

で定義される)で定義される。 A device operating in a fluid with a plurality of NEMS transducers immersed in a fluid forming an array of adjacent transducers, each NEMS transducer emitting an output signal, and two adjacent NEMS The present invention will be described as a device in which the motion of the transducers are each coupled through a fluid in which adjacent NEMS are immersed. Cross correlation of motion of first and second NEMS transducers composed of two adjacent NEMS transducers: C ₁₂ = <x ₁ (0) x ₂ (t)>

(Where k _B is the Boltzmann constant, T is the temperature of the fluid, t is the time, and X ₁₂ is the displacement x ₂ (t of the second transducer relative to the force F ₁ acting on the first transducer) ), Which results in the ensemble average of the second transducer position:

Defined in).

  The present invention comprises a microfluidic flow channel carrying a fluid flow and at least one NEMS transducer disposed in the fluid flow channel, wherein the fluid property is detected by the NEMS transducer and operates in a fluid. It is a device to do. The NEMS transducer is biofunctionalized, and the characteristics of the fluid that the NEMS transducer detects are whether the ligand that the NEMS transducer is biofunctionalized is present or absent in the fluid. It is a characteristic.

  The apparatus of the present invention further comprises a plurality of NEMS transducers, each of which is commonly arranged in the flow channel. The apparatus of the present invention further comprises a plurality of flow channels in which a plurality of NEMS transducers are distributed. The plurality of NEMS transducers are surface fabricated or filmed.

The present invention includes providing a heterostructure including a wafer layer, an etch stop layer over the wafer layer, a NEMS device layer over the etch stop layer, and a piezoresistive layer over the NEMS device layer. A method of manufacturing a bio-NEMS device from a membrane is included. Trench etching is performed through the wafer layer to the etch stop layer to define the region that will be the film from which the NEMS device is defined.
The etch stop layer is removed from the bottom of the trench to the device layer to produce a film. Conductive contacts are selectively fabricated on the piezoresistive layer of the film by electron beam lithography. A region to be biofunctionalized on the piezoresistive layer of the film is selectively formed by electron beam lithography. The NEMS device is selectively formed by electron beam lithography on the piezoresistive layer of the film including the region to be biofunctionalized. This film is selectively plasma etched to define a suspended NEMS device, except for unmasked portions. A flow channel is selectively formed in an elastomeric layer disposed around the membrane. The selected area on the NEMS device is biofunctionalized.

  Providing the heterostructure further includes polishing and thinning the wafer layer to promote adhesion with the elastomeric layer. The step of selectively plasma etching the film to define the suspended NEMS device except for the unmasked portion includes selectively removing the unmasked portion of the NEMS device layer by plasma etching vertically. Yes. The step of selectively shaping the flow channel in an elastomeric layer disposed around the membrane selectively disposes a photoresist layer for defining the flow channel and on the selectively disposed photoresist layer. Placing an elastomer layer on the substrate and then removing the photoresist layer to define a flow channel.

  The present invention provides a resonant membrane having a tip immersed in a fluid, a binding molecule attached to the tip, and a damping force that is attached to the binding molecule and dissipates noise applied to the membrane from the fluid. A NEMS device operating in a fluid comprising a fluff ball is disclosed.

  The present invention also includes a method of operating the NEMS device.

  The apparatus and method of the present invention have been and will be described in terms of action for grammatical fluency, but the claims are further defined in Section 112 of the US Patent Act. Unless otherwise stated, the limited interpretation of “means” or “step” should not necessarily be construed as limiting, the meaning of the definition provided by the claims and the full scope of equivalents judicially. All legal equivalents under 35 U.S.C. 112 if the claims are to be given explicitly under 35 U.S.C. 112 It should be understood that this should be given. Turning now to the drawings, the present invention can be better envisioned, where like elements are referred to by like numbers.

  The invention and its various embodiments will now be better understood by moving to the following detailed description of a preferred embodiment presented as a concrete example of the invention as defined in the claims. I can do it. It is hereby understood that the invention defined by the claims may be broader than the specifically claimed embodiments described below.

An exemplary embodiment is an embodiment relating to constraints on the level of current bias that can be applied to a piezoresistive bio-NEMS device in fluid. The force sensitivity that can be achieved is clearly determined by the maximum level of current bias that can be tolerated if the response is proportional to the bias current, ie R = IG. The maximum practical level of bias current depends on the maximum temperature rise of the bio-NEMS that is considered acceptable.
The bio-NEMS transducer or cantilever 10 shown for purposes of illustration in the perspective view of FIG. 12a and the top view of FIG. 12b of the micrograph can be compared as having the form of a “dive plate with a cut-off at its base”. Should be considered. However, it should be particularly understood that the form of transducer 10 is quite general and includes any type of cantilever, doubly clamped beam, paddle or any other sub-micron vibrating structure. is there. Depending on the configuration of the device 10, as shown in FIG. 12b, the fluid damping characteristics of the cantilever 16 having one or more legs 20 having a width b and depending on the length l and the width w are shown. Maximization occurs in the limiting region 12, which can increase the bending stiffness of the cantilever 16 without limitation or can be variably designed. Also, the cantilever 16 is provided with a normal electrode (not shown), which allows a normal external measurement circuit (not shown) to provide a bias current to the leg 20 when the leg 20 is bent. It should be understood that the change in can be measured. Further, the external driving force may or may not be applied to the cantilever 16 in a normal manner, depending on the application and design choice.

The preferred embodiment has two legs 20. We in the l width length is w and thickness t, the temperature rise of 1K about the biological function of the tip 14 of the resonance frequency of the reduced pressure is ω _{0/2 TT} and the cantilever 16 force constant is K It is assumed that it can withstand.

(式中、Ｐはビーム16の断面積Aの周長である)で得ることができる。

及び

(式中、k_Si=1.48x10²W/mKはシリコンの熱伝導率であり、kH₂O=0.607W/mKは水の熱伝導率である)を推定する。消散領域x<l₁では、我々は

を持っている。温度は熱フラックス(heat flux)であるからl₁にて連続しているので、x>l₁の場合、温度は単調に低下するにちがいないという境界条件を我々は持っている。 We deal with this problem as a one-dimensional problem with the heat flowing into the support substrate 18 at the support end 17 in the restricted region where the length of the beam 16 is l ₁ and the cross section is A. In the exemplary embodiment, the cantilever 16 is assumed to be made of silicon and immersed in water, although any nanomachineable material can be used and any ambient fluid can be considered. The distance x from the connecting portion 17 to the support of the cantilever 16 toward the cantilever tip 14 is measured. If x> l ₁ , a rough estimate of the heat loss for the water or fluid in which the device 10 is immersed is the relation

(Where P is the circumference of the cross-sectional area A of the beam 16).

as well as

(Where k _Si = 1.48 × 10 ² W / mK is the thermal conductivity of silicon and kH ₂ O = 0.607 W / mK is the thermal conductivity of water). In the dissipation region x <l ₁ , we

have. Since the temperature is a heat flux and is continuous at l ₁ , we have the boundary condition that if x> l ₁ the temperature must decrease monotonically.

  We consider three example devices 10 shown in Table 1. In the case of the first cantilever 16, its simple thermal conductivity calculation shows that a 1 K temperature rise at the biofunctionalized tip 14 is obtained with a steady-state bias current I = 250 μA, with a power consumption of about 10,670 μA W. Is shown. A maximum temperature rise of 12K occurs in the restricted region 12 about 2.3 μm away from the edge of the substrate 18 in the restricted region. The first device produces a response R = IG of about 8 μV / nm for this bias current.

In the case of the second cantilever 16 in Table 1, we can get the same maximum temperature increase of 12K in the restriction region 12, but this coincides with the temperature increase of 0.04K at the end 14 and occurs for a current of 75μA . For this device 10, we expect the gauge factor to be G = 5.2 × 10 ⁹ Ω / m. Therefore, the expected response is 390 μV / nm.

Finally, in the case of the third cantilever in Table 1, we can obtain a current of 22 μA using the 12 K maximum temperature rise in the restriction region 12 and the 0.04 K maximum temperature rise in the tip 14. For this device we expect G = 5.3x10 ¹⁰ Ω / m. Therefore, the expected response is 1.2 mV / nm.

In applications where the fluid coupling scaling device 10 in a bio-NEMS is driven by thermomechanical noise coupled to the fluid in the submerged fluid, this signal is relative to the Johnson noise of the device 10 background. It is advantageous to be maximized. The relative strength of these two thermal noise sources is determined by the dimensions of the cantilever 16. These dimensions depend on the magnitude of the fluid damping that determines the spectral density of the fluid-coupled thermomechanical noise in the force domain and the cantilever 16 (damping, effective mass, gauge factor, spring constant and the same calorific value at the tip. It is responsible for both the response function (depending on the current that can occur).

If the thickness t is fixed, a substantial improvement in the signal strength can be achieved by reducing the width of the cantilever leg 20, which is a measure of the signal strength of the device 10 (nV / Hz ^1/2 ) is shown in the graph of FIG. 1, which is graphed against the frequency of thermomechanical and Johnson noise in a device with widths b = 0.4 μm and b = 0.1 μm. Increasing the overall length l and width w of the cantilever 16 increases the coupling to the fluid, increases both the damping by the fluid and the cooling efficiency, and improves the S / N ratio.

  The effect when the length l of the cantilever 16 is increased is common to b = 0.1 μm, t = 130 nm and w = 5 μm in each example, and the cantilever 16 having a different length l as shown in the insertion table is used. This is shown in the graph of FIG. The ambient fluid was diethylene glycol. FIG. 2 is a graph of thermomechanical noise attenuated by the fluid, showing that attenuation increases with background Johnson noise as the cantilever length increases. When the length is 35 μm, the magnitude of the thermomechanical noise peak attenuated by the fluid is larger than the background Johnson noise. For each cantilever length, the bias current was selected so that the estimated temperature rise at tip 14 did not exceed 1 ° C and the maximum temperature of leg 20 did not exceed 50 ° C. The bias current used and the estimated temperature rise are shown in Table 2.

It should be noted that the magnitude of fluid-coupled thermomechanical noise depends on the viscosity of the fluid in addition to depending on the dimensions of the cantilever. FIG. 3 shows cantilevers 16 with b = 0.6 μm, t = 130 nm, w = 2.5 μm, l = 15 μm and l ₁ = 0.6 μm at a bias current of 250 μA for fluids of different viscosity, water, diethylene glycol, glycerin and ethylene glycol. Shows the expected signal in the voltage domain of Johnson noise and thermomechanical noise.

Increasing the probability of a receptor-ligand reaction In one embodiment, the device 10 is biofunctionalized by binding a bioreceptor molecule 24 to or near the tip 14 of the cantilever 16, as shown diagrammatically in FIG. One of the basic features of the NEMS device 10 is the use of a bioreceptor molecule 24 bound to the “functionalized” region of the elastic cantilever 16, so that the target ligand 22 for the receptor-ligand binding reaction is targeted. It is important that the rate constant (probability of reaction) is the highest value possible. However, since evolution does not select this property, it is well known that biologically expressed receptor molecules 24 generally do not have the highest possible rate constant for their target ligand. Therefore, it is effective to test various methods for increasing the binding rate constant for the target receptor 24 in the use of the NEMS device 10.

  One possible method is to use catalytic equivalents by finding specific molecules that, when closely associated with a particular species of receptor 24, reduce the activation energy of its receptor-ligand binding. It is. The rate constant and thus the probability of reaction is very sensitive to the activation energy and depends, for example, exponentially, so that the binding constant increases greatly even if this energy decrease is relatively small. Thus, the idea is first the idea of binding these layers of “catalytic” molecules 26 to the small functionalized region 28 of the cantilever 16 and then binding a specific receptor 24 to the target ligand 22 of interest. The basic concept of this method is shown in FIG. The choice of catalyst in a given example is directed by a ligand-receptor pair according to well-known principles.

  A second feasible method is to design a receptor molecule 24 whose binding rate constant is maximized for the specific ligand 22 of interest. The term “design” in this case is a forced evolution that selects a gene that expresses the desired receptor 24 with an increasingly higher binding affinity for the selected ligand 22 while “evolutioning” the gene multiple rounds. It is understood to mean the biochemical method. This is usually accomplished by inserting multiple copies of the gene for the receptor of interest into the genome of that particular bacterium so that the bacterium expresses the receptor 24 of interest on its cell surface.

  A binding affinity assay is then performed on the first generation of bacteria, and only the bacteria with the highest affinity are stored for use in the next round of the “evolution” cycle. Before starting the next cycle, various receptor genes are created by point mutation methods or more efficiently by “DNA-shuffling”. The cycle is then repeated until it becomes clear that there is no possibility of further increasing the binding affinity. By utilizing this method, it can be expected that the binding rate constant for a particular receptor-ligand can be increased by at least an order of magnitude.

Effects of Nonspecific Ligand Binding FIG. 5 shows an example of the possible effects of “background” nonspecific binding on the response of NEMS cantilever 16. Curve 30 shows the fractional occupancy of functionalized site 28 by target ligand 22, while curve 32 shows the fractional occupancy by both target 22 and background ligand molecule 32. The conditions utilized are: (1) 1000 target molecules 22; (2) 100 receptor molecules 24; and (3) 10,000 non-specific binding affinities of target ligand molecules 22 Background ligand molecule 32. The effect of nonspecific binding competition for receptor 24 is significant for the device 10 shown in FIG.

(式中、Aは周波数ω₀で発信する信号の振幅であり、そしてn(t)は分散がσ² _nであるゼロミーンガウスノイズプロセス(zero-mean Gaussian noise process)である)で表わされる。ここでAとω₀の両者が決定的であればすなわち搖動しないならば、その最適検出器はいわゆる「位相検波器」又は「ロックイン増幅器」として知られており、そのブロック図を図6に示してある。通信理論では、このような検出器を相関受信機と呼ぶ。したがって、我々は、周波数がω₀の「キャリヤ」信号を注入できて、その結果、標的リガンドの結合事象がこのキャリヤを「変調」しすなわちAの価を修正する装置の配置構成を探す。図6に示す相関検出器は、その入力r(t)を装置10からとる狭帯域フィルタ34を備えている。発信機36からの参照信号を、ミクサ38を使ってフィルタ34の出力と混合する。そのフィルタされ混合された信号を低域フィルタ40中に入力し次に閾値決定回路(thresholding and decision circuit)42に結合し、その回路は第一に前記信号を有効信号すなわち情報保有信号とみなされるかどうかを決定し、次にそのようにみなされたならば決定アルゴリズムを実行して装置10が対象のリガンド-受容体相互作用を検出したか又は検出しなかったかを決定する。これらの回路は、従来の設計選択肢によって案出されるハードウェア設計、ファームウェア又はソフトウェアによって制御されるアナログ信号又はディジタル信号のプロセッサ又はコンピュータ内で実現できる。 Using multiple cantilevers to generate a signal in bio-NEMS In the case of a single undriven cantilever used to detect biomolecules, the name of the invention is `` A METHOD AND APPARATUS FOR PROVIDING SIGNAL ANALYSIS OF A BIONEMS RESONATOR ”Is analyzed in detail in a co-pending patent application filed simultaneously with the present application under the application number (CIT.PAU.35). This patent application is incorporated herein by reference. So we analyzed the expected detection performance for some of the two driven cantilever systems. In the illustrated embodiment, we explore a number of common issues associated with using multiple cantilevers in the bio-NEMS device 10. We begin this analysis from the perspective of signal “design”, that is, we are looking for an arrangement of devices that produces an optimal signal for detection performance. Such a signal has the following formula:

(Where A is the amplitude of the signal transmitted at frequency ω ₀ and n (t) is the zero-mean Gaussian noise process with variance σ ² _n ) . If both A and ω ₀ are deterministic, i.e., do not perturb, the optimal detector is known as a so-called “phase detector” or “lock-in amplifier” and its block diagram is shown in FIG. It is shown. In communication theory, such a detector is called a correlation receiver. We can therefore inject a “carrier” signal of frequency ω ₀ , so that the binding event of the target ligand “modulates” this carrier, ie looks for an arrangement of devices that corrects the value of A. The correlation detector shown in FIG. 6 includes a narrowband filter 34 that takes its input r (t) from the device 10. The reference signal from the transmitter 36 is mixed with the output of the filter 34 using a mixer 38. The filtered and mixed signal is input into a low pass filter 40 and then coupled to a thresholding and decision circuit 42, which first considers the signal as a valid or information-carrying signal. And then, if so deemed, a decision algorithm is executed to determine whether the device 10 has detected or has not detected the ligand-receptor interaction of interest. These circuits can be implemented in a hardware design, firmware or software controlled analog or digital signal processor or computer devised by conventional design options.

  However, we must handle the relatively strict constraint that there is a severe performance loss when the signal amplitude A or phase θ is subjected to significant random fluctuations. This performance loss is shown in FIG. 7, in which case A is a “no random swing” case, case B is a case with random amplitude swing, and case C is This is a case where there is a loss of phase information due to random oscillation. In the case of labeled “Chopper”, it is the performance of a single undriven cantilever 16 that utilizes probability detection performed on a sum of multiple samples as disclosed in the above-incorporated and co-pending application. Thus, the method of injecting a carrier signal and then “binding” the signal to a ligand-receptor binding event becomes an important issue to consider in order to obtain a meaningful measurement result.

Figures 8a-8c show two possible embodiments that can generate a signal of the form of Equation 1. In FIG. 8a, carrier injection is achieved by mechanically driving an unfunctionalized cantilever 16a without a receptor at a fixed frequency ω ₀ and a fixed amplitude. The functionalized cantilever 16b responds to the driver 16a by fluid dynamic coupling of the fluid in which the device 10 is immersed. The driven cantilever 16b has a piezoresistive portion 46. Both cantilevers 16a and 16b are connected to a support 48, which in turn is connected to a substrate 44 or 50.

  In FIGS. 8a-8c, the “no signal” case is a case where the cantilever 16b is constrained from moving significantly by “tacking” it onto the substrate 44, and the “free” target ligand 22 If this is present, this state is destroyed by competitive binding. It should be noted that the parallel arrangement shown in FIG. 8a is not a preferred arrangement. In practice, it is better to have two cantilevers facing each other, i.e. the tips facing each other. FIG. 8c is a slightly different variation of FIGS. 8a and 8b, in this case having two cantilevers 16 and 34 mechanically linked by a ligand-receptor binding arrangement in a no-signal state. Again, the presence of free target ligand destroys this state by competitive binding, resulting in loss of coherent signals.

  Although we have considered multiple cantilevers 16a and 16b, we can achieve very efficient carrier injection using a single cantilever 16 plated with a magnetic thin film in a small region 28 and It should be pointed out that the external carrier signal source 36 generates a drive signal and is magnetically coupled to the cantilever to obtain a constant phase oscillation of the cantilever beam 16 with a “constant” amplitude. In fact, this method can better satisfy the perturbation of parameter perturbations than other methods. This coupling can also be extended to electrostatic coupling using a dipole film or a paraelectric film.

  A different use of the plurality of cantilevers 16 is to utilize an array of N identical non-driven cantilevers in an averaging mode or a coincidence mode. In averaging mode, we can simply use N outputs to improve our estimate of variance. In coincidence mode, we take advantage of the restriction that the fraction of N outputs must exceed the threshold within a period to be considered in a “signal present” event. In both cases, the method using multiple cantilevers aims to improve the ligand capture rate by increasing the number of available receptors 24.

Although the study of single cantilevers in fluids is quite well developed, no attention has been paid to the fluid coupling of the array of cantilevers 16. The turbulence of the fluid generated by the vibrating cantilever 16 falls off only as the inverse power of the spacing between the cantilevers 16 for a long period of time. When one cantilever 16 moves, the other cantilever 16 moves due to its viscous drag or fluid coupling between the two cantilevers. Correspondingly, there is a correlation of the stochastic motion of the cantilever 16 because the stochastic force caused by the molecular collision with one cantilever 16 induces the motion of the second cantilever 16 by the above coupling.

  This connection is made quantitatively by the oscillation dissipation theory. Correlation fluctuations induced by fluid tend to obscure the correlations induced by molecules tethered between cantilevers 16, making detection and characterization of biomolecules using this method more difficult To do. Therefore, it is important to understand the correlation induced by fluids in the absence of biomolecules and to design forms and protocols that minimize that correlation.

  Recent experiments by Meiners and Quake on spherical beads controlled by laser tweezers show for the first time expected results (JC Meiners et al., Direct Measurement Of Hydrodynamic Cross Correlations Between Two Particle In An External Potential, Phys. Rev. Lett., 82, 2211 (1999)). These two authors studied the correlation induced by the fluid interaction of two 1 μm latex beads spaced approximately 3-10 μm apart. They found a strong anticorrelation of these spheres, whose maximum anticorrelation was close to the mean square of the displacement of the single sphere at the closest distance (3 diameters) they studied. It was. This suggests that there is a strong fluid coupling that is difficult to estimate. However, since the flow around the long cantilever 16 has completely different characteristics than the flow around the sphere, this suggests a strategy for minimizing the fluid correlation.

The flow of low ray nozzles around a moving sphere is in phase with the movement of the sphere everywhere and decreases in proportion to 1 / r. Our simple model of cantilever 16 is a model that approximates a cantilever as a long cylinder compared to its radius. In this case, the Stokes test results can be used for the flow around an infinite cylinder. The flow around the oscillating cylinder is more complex than in the case of a sphere. In fact, if the value of ray nozzle number is small, there is no solution unrelated to ray nozzle number, and obvious frequency and distance between fluid velocity and cylinder velocity, changing aR ^-1/2 by several times the scale of distance There is a phase relationship that depends on. Where a is the radius of the cylinder we have the width of the cantilever and R = ωa ² / 4ν is the number of Ray nozzles, where ω is the frequency and ν is the kinematic viscosity of the fluid.

  For bio-NEMS cantilevers 16, R is generally about 1. Since this velocity region is the cause of the movement of the second cylinder, there is a distance and frequency dependency on the movement of the second cylinder induced by the force on the first cylinder. Through the general principle of perturbation-dissipation specifying that the perturbation present in the system is proportional to the attenuation in the system, this shows us the distance and frequency dependence on its noise correlation. The plot of the velocity domain as a function of the distance r / a from the cylinder axis shown in Figure 9 actually shows that different quadratures in the velocity domain have null points at different distances. Suggests that the parameters (cylinder distance / radius and frequency) corresponding to the null point of one or the other phase of the correlation noise induced by the fluid can be found. FIG. 9 shows the component of the fluid velocity parallel to the velocity of the cantilever 16 during vibration as a function of the distance r in units of the cantilever width a when the number of lay nozzles R = 1. It should be noted that the non-trivial phase relationship is given by the real (in-phase) and imaginary quadrature components and the null points of the different quadrature components.

(式中、X₁₂は第一カンチレバー16に作用する力F₁に対する第二カンチレバー16の変位x₂(t)を示す「サスセプティビリティー」である)並びに下記式：

で表わされる。 The calculation of the correlation that can optimize the design is performed as follows. The exact relationship between the cross-correlation C ₁₂ = <x ₁ (0) x ₂ (t)> between two cantilever displacements and the deterministic fluid coupling between the cantilevers is:

(Where X ₁₂ is “susceptibility” indicating the displacement x ₂ (t) of the second cantilever 16 with respect to the force F ₁ acting on the first cantilever 16) and the following formula:

It is represented by

The above square brackets mean the overall mean and emphasize that the stochastic motion can be quantified by deterministic estimation, calculation or experiment. Since the susceptibility X ₁₂ can be calculated from the Stokes velocity region plotted in FIG. 9, although not shown here, the correlation of noise can be shown.

Fusion of NEMS and Microfluidics FIG. 10 is a schematic cross-sectional view of an apparatus 10 with a cantilever 16 coupled to a microfluidic flow channel 52. The area etched through wafer 54 forms part of the final flow channel. An entire array of cantilevers 16 can be made in a single flow channel 52. Depending on the desired application, multiple channels 52 with different devices 10 in different channels 52 may be provided. It is also possible to make the flow channel 52 directly on the surface of the silicon. This is illustrated schematically in array 56 of FIGS. 11a-11c. These figures are three perspective views which are gradually enlarged, and are supported by a parallel support 48 in a single flow chamber 58 communicating with the inlet and outlet flow channels 52 of the parallel cantilever 16. A plurality of cantilevers 16 forming a row are shown.

Fabrication of Bio-NEMS Membrane Based Device FIGS. 13a-13g are flow diagrams outlining the steps involved in fabricating the membrane based bio NEMS device 10. FIG. The fabrication of these devices begins in FIG. 13a in a side sectional view showing a silicon device layer 148 on an insulating wafer 152, 154, such as a 375 nm SiO ₂ layer 152 on a 675 μm Si layer 154, for example. The buried oxide layer 152 must be thick enough to serve as a stop layer for etching steps performed through the backside of the wafer 154. The Si device layer 148 should be the desired thickness as an undoped portion of a silicon cantilever that is between 20 nm and 100 nm in thickness for most devices under consideration. In the illustrated embodiment, an 80 nm Si layer 148 is disposed below the 30 nm heavily doped Si layer 150. The resistivity of layer 148 must be high compared to the heavily doped layer 150 being grown (10 Ωcm is sufficient).

  The back surface of the wafer 154 is polished. This polishing is necessary to adhere the elastic elastomeric material to the back surface and define a microfluidic channel 52 in the elastomeric material as described below. Wafer 154 may be simultaneously thinned to help reduce the final volume of flow channel 52 and the required thickness that must be etched in a later etching step. A final wafer thickness of 300 μm is reasonable to maintain the structural integrity of the wafer 154 while reducing unnecessary material.

Next, a silicon layer 150 heavily doped with boron is epitaxially grown on the top surface of layer 148. Layer 150 forms a conductive layer of a piezoresistor that forms part of NEMS device 10. For most devices 10, the thickness of this layer 150 is between 7 nm and 30 nm. The resistivity of layer 150 must be small compared to the underlying layer 148. A doping level of 4x10 ¹⁹ cm ^-3 is common.

The next step is to manufacture the film by etching through the backside of the wafer 154 as shown in the side cross-sectional view of FIG. 13b. This step can be performed using Bosch deep reactive ion etch (DRIE) to create trench 158 through layer 154. For this step, it is sufficient to use a photoresist or oxide mask 156 of about 6 microns as shown in FIG. 13b. A 50 μm ² membrane is used, but this is optional and the dimensions used depend on the application. Next, the oxide layer 152 immediately below the silicon film 148 is removed from the bottom of the trench 158 with hydrofluoric acid to define a film 162 as shown in FIG. 13c.

  Next, photolithography and metal deposition are performed on the top surface of device 10 above layer 150 in alignment with film 162 to produce contact pad 160 as shown in the plan view of FIG. 13d. FIG. 13d shows a plurality of dies formed simultaneously. An ohmic contact is formed on the boron doped silicon layer 150 by depositing 30 nm of chromium (as an adhesion layer) followed by 250 nm of Au in the desired pattern of the bond pad 160.

  A silicon nitride layer 174 (300 nm) is then deposited over the Au, followed by a 200 nm silicon dioxide layer 175 to protect the device 10 as shown in the plan view of FIG. 13e, followed by FIG. A chromium layer 176 is deposited as shown in the plan view to protect the silicon dioxide 175 during the manufacturing process and energization is provided through the stepped height formed by these protective layers as shown in FIG. 13g.

  Then, using electron beam lithography, first pattern the gold pad (not shown) on the tip 14 where the cantilever 16 is biofunctionalized, and then pattern the cantilever 16 (on the PMMA). As shown in the perspective view of FIG. 13i, a 30 nm layer of chromium 178 is deposited and lifted off. This part of the manufacturing process involves two steps. First, there is a step of placing a gold square portion 180 (not shown in the drawing) at the tip 14 of the cantilever 16 used for biofunctionalization along an alignment mark (not shown in the drawing). The second step is a lithographic step performed to mask the area containing the cantilever 16 with the chromium layer 178 (not shown).

Except for the unmasked portion of the film defined in layers 150 and 148 by vertical plasma etching (NF ₃ , Cl ₂ , Ar), the cantilever 16 is removed as shown in FIG. Once formed, the device 10 is suspended. The wet mask is then used to remove the chromium mask 178 (not shown) and the sample is then dried in a critical point dryer. The wafer is diced as shown in FIG.

  The microfluidic channel 52 is then fabricated from a silicone elastomer using a photoresist patterned as a mold. In this case, the photoresist is then etched to create the actual flow channel defined in the elastomeric envelope 182 shaped as shown in FIG. 13i. The flow channel 52 self-seals with the silicon of the device 10 when placed in an 85 ° C. oven for 24 hours. The device 10 or gold pad 180 is then biofunctionalized by conventional means as shown in FIG. 13m by flowing fluid through the flow channel 52, for example, carrying a receptor molecule that preferentially binds to the gold pad 180. To do.

Attenuation by fluff ball A fluff ball 60, which is only a large dissipation molecule, is associated with a binding receptor molecule 62. The fluff ball 60 with the binding molecule 62 floats freely in the liquid. The binding molecule 62 is configured to bind to the ligand of interest. The tip 14 is also biofunctionalized with a receptor configured to bind to the ligand of interest. When the receptor at the tip 14 captures the ligand of interest at the bound receptor 62 and fluff ball 60, the extinction coefficient of the tip 14 increases dramatically.

(式中、Mはカンチレバー16の質量であり、Yはカンチレバー16の流体による減衰係数であり、kはカンチレバー16のばね定数であり、xはカンチレバー先端14の変位であり、k_mは分子62の有効ばね定数でありそしてｘ_dはフラフボール60の変位であり、Y_dはフラフボール60の流体による減衰係数でありそしてFはカンチレバー16に加えられる外力である)である。 FIG. 14 is a mathematical model of a mass M cantilever 16 having a fluff ball 60 connected to the tip 1 by a molecule 62 treated as a spring. The fluff ball 60 is devised to maximize noise in accordance with maximum dissipation, and is composed of a star dendrimer. Molecule 62 is composed of a chain of alkanes or ligands. The system equation shown in FIG.

(Where M is the mass of the cantilever 16, Y is the damping coefficient of the cantilever 16 due to the fluid, k is the spring constant of the cantilever 16, x is the displacement of the cantilever tip 14, and k _m is the numerator 62 And x _d is the displacement of the fluff ball 60, Y _d is the damping coefficient due to the fluid of the fluff ball 60, and F is the external force applied to the cantilever 16).

で表わすことができる。フラフボール60は、散逸を最大にするためしたがってノイズも最大にするため減衰係数をできるだけ大きくするように下記式：

のように選択される。 The equation of motion for this system has been rewritten to show that there is an effective damping constant and effective spring constant for the system:

It can be expressed as The fluff ball 60 has the following formula to maximize the attenuation coefficient to maximize dissipation and hence noise:

Is selected.

の程度である。 The fractional dissipative effect on the dissipation and noise of fluffballs with biomolecules is:

It is the degree.

  Many alterations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it should be understood that the embodiments specifically described herein are set forth by way of illustration only and should not be taken as limiting the invention as defined by the appended claims. I must. For example, despite the fact that multiple elements of a claim are described in a particular combination, the invention may be applied to fewer elements, more elements, or other combinations of different elements. It must be seriously understood that any combination disclosed above is intended to be included even if such combinations are not initially claimed.

  The terms used in this specification to describe the invention and its various embodiments are defined not only in their generally defined meanings, but also by their special definitions in this specification. It should be understood to include structures, materials or functions beyond the meaning of the meaning. Thus, if an element can be understood to encompass more than one meaning within the context of this specification, its use in the claims is intended to be all that is supported by this specification and the term itself. Must be understood to be inclusive.

  Accordingly, the definitions of the words and elements in the appended claims do not only have the literal combinations of elements described in the specification, but also operate in substantially the same manner. Defined as encompassing all equivalent structures, materials or functions that achieve substantially the same result. Thus, in this sense, any one element in the above claims may be equivalently replaced by two or more elements, or two or more elements in a claim may be replaced by a single element. The author intends that it can be replaced with. Although more than one element may have been described above and may be originally claimed to work in a particular combination, one or more elements from the claimed combination may, in some cases, be included in that combination. It should be understood that the claimed combinations may be directed to partial combinations or variations of partial combinations.

  From the perspective of those skilled in the art, it is intentionally intended that insubstantial changes from the claimed subject matter, whether currently known or devised in the future, are equivalent within the scope of the claims. Yes. Thus, obvious substitutions now known or later known to those skilled in the art are defined to be within the scope of the defined elements.

  Therefore, the scope of the claims includes what has been specifically illustrated and described above, what is conceptually equivalent, what can be clearly replaced, and what essentially incorporates the essential idea of the present invention. It should be understood as including.

All other dimensions are fixed and are graphs of the observed improvement in thermomechanical noise intensity with respect to Johnson noise coupled to the fluid. 2 is a graph of thermomechanical noise attenuated by a fluid. FIG. 6 is a graph of signals expected in a number of liquids for a cantilever bias current of 250 μA for b = 0.6 μm, t = 130 nm, w = 2.5 μm, l = 15 μm and l ₁ = 0.6 μm. It is a side view of the cantilever expanded microscopically. Figure 5 is a graph of fractional receptor occupancy as a function of non-specific binding events. 1 is a diagram of a detector known as a “phase detector” or “lock-in amplifier” or correlation receiver. FIG. Fig. 5 is a graph showing detector performance Pd as a function of SNR. FIG. 2 is a top view of a double cantilever system. FIG. 8b is a cross-sectional side view of the system shown in FIG. 8a. FIG. 6 is a plan view of another embodiment in which the cantilever is bound with a ligand. FIG. 6 is a graph of the velocity component of a fluid parallel to a vibrating cantilever as a function of distance r. 1 is a schematic cross-sectional side view of a piezoresistive cantilever coupled to a microfluidic flow channel. FIG. It is the perspective view which increased and showed the magnification of the array of the cantilever shown in FIG. It is a photograph of the scanning electron microscope of a bio NEMS converter. Fig. 12b is a top view of the converter shown in Fig. 12a. FIG. 3 is a series of diagrams illustrating a method of manufacturing a bio NEMS fluid sensor from a membrane. FIG. 3 is a diagram illustrating the dynamic operation of a cantilever with molecularly coupled fluff balls as additional dampers.

Claims (34)

A support, and a piezoresistive cantilever having a length l, a width w and a tip coupled to and extending from the support, and having a narrow width b and a length l ₁ A piezoresistive cantilever having a limiting portion and a biofunctionalized portion at or near the tip,
A sub-micron bio NEMS device. 2. The bio-NEMS device according to claim 1, wherein the restriction portion is composed of a plurality of legs having a width b which is coupled to the support and has a narrow width. The bio-NEMS device according to claim 1, wherein the plurality of legs are two legs separated from each other by a distance w-2b. The bio-NEMS device according to claim 1, further comprising a power supply for bias current applied to the limiting portion of the cantilever, and wherein the magnitude of the bias current is limited by the maximum temperature rise allowed at the biofunctionalized tip. . The bio-NEMS device according to claim 4, wherein the maximum temperature rise allowed at the biofunctionalized tip is about 1K. In a fluid comprising at least one vibrating cantilever of length l and having a size selected to minimize background Johnson noise associated with the signal intensity produced by the piezoresistive bio-NEMS device Improvement of piezoresistive bio-NEMS device immersed in water. The improvement of claim 6, wherein the signal strength is based on the level of thermomechanical noise of the piezoresistive bio-NEMS device in the fluid. A piezoresistive bio-NEMS device, wherein the piezoresistive cantilever is w and has a narrow width b limit, the narrow width b being selected to reduce Johnson noise related to the signal intensity emitted by the piezoresistive bioNEMS The improvement of claim 8, wherein the signal strength is based on the level of thermomechanical noise of the piezoresistive bio NEMS device in the fluid. Fluid comprising a receptor disposed on a bio-NEMS device for binding to a target ligand and a catalyst disposed on the bio-NEMS device with the receptor to increase the binding rate coefficient of the receptor to the target ligand Improvement of bio-functionalized bio-NEMS device immersed in the inside. 11. The improvement of claim 10, wherein the catalyst reduces the receptor-ligand binding activation energy. The improvement according to claim 10, wherein the receptor is designed by forced evolution to bind preferentially to the ligand of interest. Carrier signal source,
Support,
A piezoresistive cantilever coupled to and extending from the support, and disposed on the cantilever and electromagnetically coupled to the carrier signal source such that the cantilever is driven by a carrier signal from the carrier signal source; Elements,
Submicron device comprising: 14. The apparatus of claim 13, wherein the element includes a magnetic film disposed on the cantilever and the carrier signal source emits an electromagnetic signal coupled to the magnetic film. A plurality of NEMS converters each emitting an output signal, and means for processing a plurality of corresponding output signals from the plurality of NEMS converters to obtain a total output signal;
A device comprising: The apparatus of claim 15, wherein the means averages a plurality of output signals such that the total output signal is an average value. 16. The apparatus of claim 15, wherein the means checks whether a predetermined fraction of the plurality of output signals exceeds a threshold within a predetermined period. Multiple NEMS transducers are each biofunctionalized, and the means effectively increases the ligand capture rate simply by issuing a comprehensive output signal indicating an increased ligand capture rate compared to one of these NEMS transducers The apparatus of claim 15. With multiple NEMS transducers immersed in the fluid forming an array of adjacent transducers, each NEMS transducer emitting an output signal, and the movement of two adjacent NEMS transducers A device immersed in fluid that is coupled together through the fluid being immersed in the vessel. Cross-correlation of motion of first and second NEMS transducers composed of two adjacent NEMS transducers: C ₁₂ = <x ₁ (0) x ₂ (t)>

(Where k _B is the Boltzmann constant, T is the temperature of the fluid, t is the time, and X ₁₂ is the displacement x ₂ (t of the second transducer relative to the force F ₁ acting on the first transducer) As a result, the overall average position of the second converter is expressed by the following formula:

The apparatus of claim 19, defined by A microfluidic flow channel carrying the fluid flow, and at least one NEMS transducer disposed in the microfluidic flow channel so as to detect the characteristics of the fluid with the NEMS transducer;
A device immersed in a fluid comprising: The NEMS transducer is biofunctionalized and the property of the fluid detected by the NEMS transducer is a characteristic of whether the ligand that the NEMS transducer is biofunctionalized is present or absent in the fluid The apparatus of claim 21. The apparatus according to claim 21, further comprising a plurality of NEMS converters, each NEMS converter being commonly arranged in the flow channel. 24. The apparatus of claim 23, further comprising a plurality of flow channels in which a plurality of NEMS transducers are distributed. 24. The apparatus of claim 23, wherein a plurality of NEMS transducers are formed on the surface. 24. The apparatus of claim 23, wherein the plurality of NEMS transducers are formed with a film. Providing a heterostructure having a wafer layer, an etch stop layer on the wafer layer, a NEMS device layer on the etch stop layer, and a piezoresistive layer on the NEMS device layer;
Perform trench etching through the wafer layer to the etch stop layer, and define a region that becomes a film in which the NEMS device is defined,
Form the film by removing the etching stop layer at the bottom of the trench up to the device layer,
A conductive contact is selectively formed by electron beam lithography on the piezoresistive layer of the film,
A region to be biofunctionalized is selectively formed by electron beam lithography on the piezoresistive layer of the film,
A NEMS device is selectively formed by electron beam lithography on the piezoresistive layer of the membrane including the region to be biofunctionalized;
The membrane is selectively plasma etched to define a suspended NEMS device, except for the unmasked part,
Selectively shaping flow channels in an elastomeric layer disposed around the membrane;
Make selected areas of the NEMS device biologically functional,
A method of manufacturing a bio-NEMS device from a membrane comprising steps. 28. The method of claim 27, wherein providing the heterostructure further comprises polishing the wafer layer to promote adhesion to the elastomeric layer. 28. The method of claim 27, wherein providing the heterostructure further comprises thinning the wafer layer. The step of selectively plasma etching the film to define the suspended NEMS device except for the unmasked portion includes selectively vertically etching the unmasked portion of the layer of the NEMS device. 28. The method of claim 27. Selectively forming a flow channel in an elastomer layer disposed around the membrane selectively disposing a photoresist layer to define the flow channel; 28. The method of claim 27 including the step of disposing an elastomeric layer thereon and then defining a flow channel excluding the photoresist layer. A resonant member having a tip that is biofunctionalized for the ligand and immersed in the fluid;
A binding molecule disposed in the fluid, and a fluff ball disposed in the fluid to provide damping force,
Wherein the capture of the ligand by the binding molecule and the capture of the fluff ball by the binding molecule increases the attenuation of the member when the ligand is bound to the biofunctionalized tip of the member. NEMS device immersed in fluid for detection. 33. The NEMS device according to claim 32, wherein the fluff ball is made of a star dendrimer. Dip the tip of the resonant member that is biofunctionalized with respect to the ligand in the fluid,
Put the binding molecule in the fluid,
Put the fluff ball in the fluid,
Capture the ligand by its binding molecule,
Capture the fluff ball by the binding molecule, and then bind the ligand to the biofunctionalized tip of the resonant member to increase the damping of the resonant member;
A method for detecting a ligand in a fluid by means of a NEMS device including a step.

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