CN100535649C - Microelectrode biosensing chip of 3D nanogap mesh array - Google Patents
Microelectrode biosensing chip of 3D nanogap mesh array Download PDFInfo
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- CN100535649C CN100535649C CNB200610066541XA CN200610066541A CN100535649C CN 100535649 C CN100535649 C CN 100535649C CN B200610066541X A CNB200610066541X A CN B200610066541XA CN 200610066541 A CN200610066541 A CN 200610066541A CN 100535649 C CN100535649 C CN 100535649C
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Abstract
The present invention relates to micro analysis chip technology, and is especially one kind of 3D micro electrode biosensing chip with nanogap mesh array. The chip has one insulating substrate, one group of gold electrodes on the substrate, one insulating material film in nanometer thickness to cover the gold electrodes and one group of electrodes perpendicular to the gold electrodes to constitute a mesh structure. The insulating layer in and around the crossing sites is eliminated to form micro flow ponds for biological sample analysis, and one large sample analyzing pond is formed with polymer on the chip. The electrode arrays in two planes may be combined in different forms to generate required electric field, and the upper and the lower electrodes constitute measuring sensors with nanometer interval. The present invention may be used in separable and addressable detection of biological sample.
Description
Technical field
The present invention relates to micro-nano biosensor technology field, is the structural design of a kind of miniature analysis chip system, can carry out particularly that the biological sample dielectrophoresis separates and a kind of microelectrode biosensing chip of 3 D nanogap mesh array of impedance detection.
Background technology
Reduce cost, the minimizing time is one of major issue of being concerned about in the clinical diagnose process, particularly for the diagnosis of molecule, cellular level.With the sensing technology is " the on-the-spot detection " (point of care testing-POCT) technology that base growth is got up, and can be described as one of current medical test circle in the world technology with fastest developing speed.Cheap, fast, portability, multifunction be the important development trend of POCT equipment, this just needs sensitivity, the micro-nano sensor support of response, Highgrade integration fast.Along with the development of infotech, biotechnology and nanometer technology, the achievement in research in these fields is constantly absorbed by the novel micro nanometer sensor, has facilitated with the major technological breakthrough headed by the biology sensor.
After the dielectrophoresis phenomenon is applied to separation of particles, shown the superiority that it is huge gradually.Compare with conventional art, at aspects such as isolation resolution, product purity, sample size, device cost and portability breakthrough raising has been arranged all based on the cell separation device of dielectrophoresis technology.At present, dielectrophoresis technology has been widely used in the manipulation and the detection of micro/nano-scale particulate, comprises seizure, the sorting of cell, virus, bacterium and DNA and separates fields such as detection.Because the widespread use of dielectrophoresis technology, a lot of biological sample treatment technologies on albumen or cellular level continue to bring out.Make a general survey of domestic and international present Research, realize that the dielectrophoresis on albumen or the cellular level separates the two kinds of approach that mainly contain: the one, utilize the velocity contrast between different particulates to make it to come along device isolation.The separation principle of this method exactly likes electrophoresis, and only the particulate of being handled can be a neutral particle.Realize the speed separation of particulate, usually will be by fluid.The 2nd, by " trap " target particle is subside from the suspending liquid that has mixed multiple particle.Utilize trap can realize the focusing of particulate, seizure and location, even can realize single molecule manipulation.This be dielectrophoresis exclusive advantage, it makes the single control and the trace detection of particle become possibility.
Bio-impedance spectrum detection technique has had in biomedicine more widely to be used, and integrated this technology has great importance in micro-system.The impedance spectrum biochemistry detection technology of combining nano technology particularly, the application in micro-system has more vast potential for future development.Studies show that in a large number the impedance spectrum that adopts microelectrode array to carry out is measured and not only can be distinguished cell category, can also reflect growth, migration and some physiological properties of cell.Yet, be feasible although utilize microelectrode array pair cell or Bacteria Detection, its measurement sensitivity is still waiting to improve.Detection sensitivity is lower, and the one, because electrode design is a planar structure, weak output signal; The 2nd, because electrode polarization produces parasitic disturbances, flood useful signal.The detecting electrode that adopts nanostructured is one of effective way that solves measurement sensitivity.Recently, people such as the Mingqiang Yi of UC Berkeley are by theory and evidence, and when the spacing of capacitor plate during much smaller than the thickness of pole plate electrostatic double layer, the influence that electrode polarization produces just can be ignored.In addition, adopt three-dimensional structure, also can improve detection sensitivity.They have designed a kind of capacitive biometric sensor with nano gap, and the device gap is 20nm, only just can detect the ssDNA of 100nM with the liquid of 1.2pl.
The data processing that develops into biochemistry detection of infotech provides the instrument of easy high speed.Since biochip technology was born, biochemical device was tight day by day with combining of computing machine, and " array " form of data collection point is to realize the fundamental way of this combination.The array format of so-called data collection point is arranged data collection point exactly according to the form of row and column." array " form of employing helps the dynamic control of standard of linear actuating means and the quick high automation read-write of computing machine.
Above-mentioned technology is for being quick on the draw, responding fast, being easy to program control microsensor wide development space is provided.But, at present also do not see relevant report in the micro-nano biology sensor research at home and abroad that above-mentioned principle and technology are combined.
Summary of the invention
The object of the present invention is to provide a kind of microelectrode biosensing chip of 3 D nanogap mesh array, the dielectrophoresis that this bio-sensing chip can be implemented on albumen or the cellular level separates and unmarked, addressable detection, and it has adopted the three-dimensional structure of nano gap to be particularly suitable for the trace biology sample is carried out the impedance measurement of rapid sensitive.
For achieving the above object, technical solution of the present invention provides 1, a kind of microelectrode biosensing chip of 3 D nanogap mesh array, comprises insulating substrate, metal microelectrode, insulation course and sample cell; It is characterized in that, at the upper surface of an insulating substrate, affixed one group of bottom electrode array that the metal microelectrode that is arranged in parallel is formed, upper surface at the bottom electrode array, an affixed insulation course, at the insulation course upper surface, affixed again second group of top electrode array that the metal microelectrode that is arranged in parallel is formed; The parallel direction of bottom electrode array, with the parallel direction perpendicular quadrature of top electrode array, upper and lower electrod-array constitutes three dimensional network structure;
Around the upper surface of top electrode array, surround a sample point sample pond with polymer projection, the area in sample point sample pond is greater than the area of upper and lower electrod-array;
Each electrode crossing place at upper and lower electrod-array, in insulation course, point of crossing with infall is that the specific region that the center surrounds is provided with the miniature pond of biological sample analysis, and the bottom surface of analyzing miniature pond is the upper surface of insulating substrate, and the miniature pond of several biological sample analysises is arranged into an array; Analyze on the top center line in miniature pond and expose top electrode, expose bottom electrode at the bottom of the pond on the center line, the orthogonal projection of upper and lower electrode at the bottom of the pond is perpendicular quadrature; Bottom, sample point sample pond interlinks with the miniature pond of analysis.
Described microelectrode biosensing chip, its described insulating substrate is silicon or glass.
Described microelectrode biosensing chip, its described metal microelectrode is gold electrode, and its shape is strip or interdigitated, and width is uniform or heterogeneous;
The upper and lower electrod-array that metal microelectrode constitutes has the dual-use function as separate mesh electrode and detecting electrode concurrently; And press row and column by peripheral addressing; In each right-angled intersection place of upper and lower electrod-array, all be the data collection point of impedance analysis; The upper and lower electrode in the point of crossing of each infall constitutes the nano gap measuring sensors.
Described microelectrode biosensing chip, its described insulation course is polymeric material or silicon dioxide, its thickness is that micron is to nanometer scale.
Described microelectrode biosensing chip, its described insulation course, the upper surface exposed section is coated with metal film, and the metal film and the metal microelectrode of covering are insulated from each other, link to each other with external circuit by lead-in wire.
Described microelectrode biosensing chip, the metal film that its described insulation course upper surface exposed section covers is golden film.
Described microelectrode biosensing chip, its miniature pond of described analysis is the place of seizure and collection of biological particulate, its shape is circular, square or rectangular; Analyze miniature pond and communicate, analyze at the bottom of the pond in miniature pond and pool wall all is exposed in the sample solution with whole sample point sample pond.
Described microelectrode biosensing chip, its described biological sample comprises cell, virus, bacterium, protein macromolecule and DNA suspending liquid; It is included in the separation on cell, virus, bacterium, protein macromolecule and the dna level to the separation on albumen or the cellular level.
The fusion that the present invention is based on dielectrophoresis principle and impedance measurement principle to the separation and the detection of biological sample.Dielectrophoresis, be exactly neutral corpuscle under the effect of non-homogeneous direct current in space or AC field, polarization taking place and the direction that increases or reduce along field intensity is subjected to clean power, and carries out the phenomenon of drift motion in liquid.Dielectrophoretic force is proportional to the gradient of field intensity mould value square, the positive and negative frequency that depends on particle species and AC signal of scale factor.Suitably select the frequency size, can make certain particulate trend towards the field intensity extreme point, and other particulates are away from the field intensity extreme point.The sensing chip that the present invention is designed, select by suitable frequency, the AC field that microelectrode array produces can make the dielectrophoresis particulate optionally " subside " in miniature pond, thereby the specific inductive capacity of crack electric capacity received that makes that upper and lower electrode forms changes in right-angled intersection place.By just can determine the physiological status of biological specimen to the impedance measurement of upper and lower electrode.
Description of drawings
Fig. 1 is the plan structure figure of microelectrode biosensing chip of 3 D nanogap mesh array of the present invention;
Fig. 2 is the sectional structure chart of microelectrode biosensing chip of 3 D nanogap mesh array of the present invention.
Embodiment
As shown in Figure 1 and Figure 2, the structural drawing of microelectrode biosensing chip of 3 D nanogap mesh array of the present invention, upper surface at an insulating substrate 1, affixed one group of metal microelectrode array that is arranged in parallel is bottom electrode array 6, at the upper surface of bottom electrode array 6, an affixed insulation course 4, insulation course 4 upper surfaces, affixed again second group of metal microelectrode array that is arranged in parallel is top electrode array 2; The parallel direction of first group of metal microelectrode array, with the parallel direction perpendicular quadrature of second group of metal microelectrode array, second group of metal microelectrode array is positioned at the top of first group of metal microelectrode array, and upper and lower electrod- array 2,6 constitutes three dimensional network structure.
At the upper surface of metal microelectrode 2 arrays, an affixed again polymeric layer 31, the area of polymeric layer 31 is greater than the area of upper and lower electrod-array 2,6.The middle part of polymeric layer 31 is removed, expose top electrode array 2, and the upper surface of insulation course 4, the polymer projection that stays all around surrounds a sample point sample pond 3.
Again with each electrode crossing place of upper and lower electrod- array 2,6, reach with the point of crossing of infall is the specific region institute covered dielectric layer 4 on every side at center, all remove, form the miniature pond 5 of several biological sample analysises, the miniature pond of several biological sample analysises 5 is arranged into an array, analyze on the top center line in miniature pond 5 and expose top electrode, expose bottom electrode at the bottom of the pond on the center line, the orthogonal projection of upper and lower electrode at the bottom of the pond is perpendicular quadrature.3 bottoms, sample point sample pond interlink with the miniature pond 5 of analysis.
Wherein, the insulating substrate material is silicon or glass.
Metal microelectrode is a gold electrode, and its shape can be strip or interdigitated, and width is uniform or heterogeneous.The size of metal microelectrode, arranging density and electrode width can consider all that with the ratio of adjacent electrode separation different operating requires specifically to determine.
The upper and lower electrod- array 2,6 that metal microelectrode constitutes has the dual-use function as separate mesh electrode and detecting electrode concurrently; And can be by row and column by peripheral addressing.In upper and lower electrod- array 2,6 each right-angled intersections place, all be the data collection point of impedance analysis; The upper and lower electrode in the point of crossing of each infall constitutes nano gap capacity measurement sensor.
Insulation course 4 is a polymeric material, and its thickness is nanometer scale, can specifically determine according to the biological specimen feature.Insulation course 4 upper surface exposed sections can cover the sheet metal of arbitrary shape, and the sheet metal and the metal microelectrode of covering are insulated from each other, are gold plaque, can link to each other with external circuit by lead-in wire.
Analyzing miniature pond 5 is places of seizure and collection of biological particulate, and its shape can be circular, square or rectangular, and its size, size and spatial arrangement all can specifically be determined according to measurement requirement.Analyze miniature pond 5 and communicate, analyze at the bottom of the pond in miniature pond 5 and pool wall all is exposed in the sample solution with whole big sample point sample pond 3.
Microelectrode biosensing chip of 3 D nanogap mesh array of the present invention, used biological sample comprises cell, virus, bacterium, protein macromolecule and DNA suspending liquid.It is included in the separation on cell, virus, bacterium, protein macromolecule and the dna level to the separation on albumen or the cellular level.
Be a specific embodiment of the present invention below:
At 1 * 1cm
2Glass substrate 1 on utilize and peel off the gold electrode array that (lift-off) technology is produced 30 μ m live widths, it is bottom electrode array 6, on the glass sheet of making bottom electrode array 6, get rid of the Kapton insulation course 4 that last layer thickness is 100 nanometer thickness again, then with peeling off the top electrode array 2 that (lift-off) technology is produced 10 μ m live widths, get rid of the thick SU8 glue 31 of 2 μ m again, carve wall thickness 500 μ m, aperture area 0.5 * 0.5cm
2Sample point sample pond 3, adopt reactive ion etching to carve aperture area 50 * 70 μ m at last
2The miniature pond 5 of sample analysis.
Claims (9)
1, a kind of microelectrode biosensing chip of 3 D nanogap mesh array comprises insulating substrate, metal microelectrode, insulation course and sample cell; It is characterized in that, at the upper surface of an insulating substrate, affixed one group of bottom electrode array that the metal microelectrode that is arranged in parallel is formed, upper surface at the bottom electrode array, an affixed insulation course, at the insulation course upper surface, affixed again second group of top electrode array that the metal microelectrode that is arranged in parallel is formed; The parallel direction of bottom electrode array, with the parallel direction perpendicular quadrature of top electrode array, upper and lower electrod-array constitutes three dimensional network structure;
Around the upper surface of top electrode array, surround a sample point sample pond with polymer projection, the area in sample point sample pond is greater than the area of upper and lower electrod-array;
Each electrode crossing place at upper and lower electrod-array, in insulation course, point of crossing with infall is that the specific region that the center surrounds is provided with the miniature pond of biological sample analysis, and the bottom surface of analyzing miniature pond is the upper surface of insulating substrate, and the miniature pond of several biological sample analysises is arranged into an array; Analyze on the top center line in miniature pond and expose top electrode, expose bottom electrode at the bottom of the pond on the center line, the orthogonal projection of upper and lower electrode at the bottom of the pond is perpendicular quadrature; Bottom, sample point sample pond interlinks with the miniature pond of analysis.
2, microelectrode biosensing chip as claimed in claim 1 is characterized in that, described insulating substrate is silicon or glass.
3, microelectrode biosensing chip as claimed in claim 1 is characterized in that, described metal microelectrode is gold electrode, and its shape is strip or interdigitated;
The upper and lower electrod-array that metal microelectrode constitutes has the dual-use function as separate mesh electrode and detecting electrode concurrently; And press row and column by peripheral addressing; In each right-angled intersection place of upper and lower electrod-array, all be the data collection point of impedance analysis; The upper and lower electrode in the point of crossing of each infall constitutes the nano gap measuring sensors.
4, microelectrode biosensing chip as claimed in claim 1 is characterized in that, described insulation course is polymeric material or silicon dioxide, and its thickness is nanometer scale.
5, as claim 1 or 4 described microelectrode biosensing chips, it is characterized in that, described insulation course, the upper surface exposed section is coated with metal film, and the metal film and the metal microelectrode of covering are insulated from each other, link to each other with external circuit by lead-in wire.
6, microelectrode biosensing chip as claimed in claim 5 is characterized in that, the metal film that described insulation course upper surface exposed section covers is golden film.
7, microelectrode biosensing chip as claimed in claim 1 is characterized in that, the miniature pond of described analysis is the place of seizure and collection of biological particulate, and its shape is circular, square; Analyze miniature pond and communicate, analyze at the bottom of the pond in miniature pond and pool wall all is exposed in the sample solution with whole sample point sample pond.
8, microelectrode biosensing chip as claimed in claim 7 is characterized in that, the miniature pond of described analysis, and its shape is rectangular.
9, microelectrode biosensing chip as claimed in claim 1 is characterized in that, described biological sample comprises cell, virus, bacterium, protein macromolecule or DNA suspending liquid; It is included in the separation on cell, virus, bacterium, protein macromolecule or the dna level to the separation on albumen or the cellular level.
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