CN1176220C - Gene chip with hairpin-shaped probes and its preparing process and detecting method - Google Patents
Gene chip with hairpin-shaped probes and its preparing process and detecting method Download PDFInfo
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- CN1176220C CN1176220C CNB021163022A CN02116302A CN1176220C CN 1176220 C CN1176220 C CN 1176220C CN B021163022 A CNB021163022 A CN B021163022A CN 02116302 A CN02116302 A CN 02116302A CN 1176220 C CN1176220 C CN 1176220C
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Abstract
The present invention relates to a gene chip which comprises a chip base and an oligomer nucleic acid probe solidified on the chip base, wherein the probe comprises a detecting zone and a stalk zone; an oligomer nucleic acid sequence in the stalk zone and the matched oligomer nucleic acid sequence form a barrette-shaped double-chain structure. The match mode and the base number of the nucleic acid satisfy the following conditions that the melting temperature of the barrette-shaped oligomer nucleic acid probe is in a range between the melting temperature of-5 DEG C of a single point mismatch hybridization state and the melting temperature of +5 DEG C of a completely matched hybridization state. The present invention can further optimize the hybridization condition by controlling the electric potential of the gene chip. The gene chip of the present invention and a sample do not need to be marked, and the chip can be repeatedly used to greatly reduce the manufacture cost and the use cost. The present invention has the capability of identifying the sequence of the oligomer nucleic acid sequence with the complete match and the single point mismatch and can be widely applied to the biotechnology field.
Description
Technical field: the present invention relates to biological technical field, relate in particular to a kind of gene chip, and its preparation method and utilize this gene chip to carry out the method for gene test.
Background technology: the research relevant with gene is a key areas in the modern life science research.Finishing of the Human Genome Project and other gene sequencing plans, for life, research that medical science is relevant, as the exploitation of the diagnosis of genetically engineered, disease and treatment, new drug, lay a good foundation.For these follow-up work, need to develop new biotechnology simultaneously, gene chip is exactly one of them example.
One type of gene chip is that the nucleic acid oligomer of known array (below be called probe) is solidified from the teeth outwards, by hybrid experiment, detect the base sequence (qualitative analysis) of the nucleic acid oligomer (gene fragment) in the sample or detect the content (quantitative analysis) of some nucleic acid oligomer base sequence.
The method that detects comprises fluorescence, electrochemistry, alternating-current impedance, QCM (Quartz Crystal Microbalance), isotopic labeling, surface plasma body resonant vibration, mass spectrum, enzyme linked immunosorbent assay etc.Wherein using maximum is fluorescence detection method.
All detection methods can be divided into two classes: a class need be carried out mark to probe or sample, as fluorescence, isotopic labeling, enzyme linked immunosorbent assay, is called underlined method; Another kind of the needs carried out mark to probe or sample, as alternating-current impedance, QCM (Quartz Crystal Microbalance), surface plasma body resonant vibration, mass spectroscopy, is called unmarked method.Some detection method can be designed to underlined method and unmarked method respectively, as electrochemical method.
1996, Tyagi and Kramer[see document S.Tyagi, and F.R.Kramer, NatureBiotechnology, 14:303 (1996)] propose to have hair fastener shape structure, made the testing tool of the probe (being called molecular beacon) of fluorescence and quenching group mark simultaneously as DNA hybridization.The design of this novelty causes gazing at of common people immediately, obtains promotion and application rapidly in biotechnology (as PCR).
Molecular beacon is being cured to the surface aspect the research that is applied to the DNA transmitter, the someone has carried out good try and [has seen document J.Li, et al., Ana.Sci.17:1149 (2001); X.J.Liu, and W.H.Tan, Ana.Chem.71:5054 (1999); W.H.Tan, et al., Chemistry-a European Journal, 6:1107 (2000); X.H.Fang, and W.H.Tan, Ana.Chem.71:3101 (1999); X.H.Fang, et al., J.Am.Chem.Soc.121:2921 (1999)].But, the expensive shortcoming of fluorescence labeling method before molecular beacon not only can not overcome, and cost of manufacture is more increased.
Nineteen ninety-five, people such as Linford and Chidsey [sees document M.R.Linford, et al., J.Am.Chem.Soc.117:3145 (1995)] find the addition reaction of Si-H key and C=C pair of keys of organic long-chain end points that can be by adding the thermal initiation monocrystalline silicon surface, form stable, fine and close organic membrane at silicon face, and found afterwards [to see document P.Wagner, et al.J.Struct.Biol.119:189 (1997), T.Strother, et.al.Nuclear AcidsResearch, 28:3535 (2000); T.Strother, et al., J.Am.Chem.Soc.122:1205 (2000)] ultraviolet lighting can be used for preparing organic membrane equally, and then nucleic acid oligomer can be connected on the organic membrane of silicon face.After this, many researchs about silicon face organic membrane character are arranged, [originally the work of first contriver participation is seen document T.Strother, et al., J.Am.Chem.Soc.122:1205 (2000) in some researchs that comprise this first contriver participation and be engaged in; Wu Ruige etc., Acta PhySico-Chimica Sinica 17:931 (2001)].
Also have the method for measuring AC impedance in the unmarked method detection technique of surperficial nucleic acid oligomer hybridization [to see document E.Souteyrand, et al., J.Phys.Chem.B 101:2980 (1997); H.Bemey, et al., Sensorsand Actuators B 68:100 (2000); C.Berggren, et al., Electroanalysis 11:156 (1999)].What this method was measured is the change of nucleic acid oligomer hybridization front and rear surfaces dielectric properties, and measuring process is simple, mild condition, and it is low to measure cost, can not damage chip, and not need nucleic acid oligomer is carried out any mark.
Though had company to produce commercial gene chip at present, these products all exist significant disadvantages: cost costliness, poor repeatability, poor stability.
Particularly, at present the problem of a common essence of all biochip technologies is poor specificity: how in detection only differentiated two the nucleic acid oligomer sequences of base on a site (two nucleic acid oligomer sequences of promptly so-called coupling fully and single-point mispairing) differentiate, be a technical barrier.
Summary of the invention:
The object of the present invention is to provide and a kind ofly can realize that the identification of simple point mutation in the nucleic acid oligomer sequence, cost are low, good stability, can reusable gene chip.
Another object of the present invention also is to provide a kind of preparation said gene chip production method.
The present invention also aims to provide a kind of method of utilizing the said gene chip to carry out gene test.
Gene chip with hairpin-shaped probes of the present invention comprises chip base and solidifies thereon nucleic acid oligomer probe to have at least a nucleic acid oligomer probe to comprise detecting area, the nucleic acid oligomer sequence that exists one section or plurality of sections to be determined by the gene order of detected sample; The cane district, the nucleic acid oligomer sequence that has a section or plurality of sections nucleic acid oligomer sequence and be complementary, the nucleic acid oligomer sequence in the cane district and form hair fastener shape duplex structure with the nucleic acid oligomer sequence of its coupling with this sequence.The matching way of the nucleic acid in cane district and base number satisfy following condition: the melting temperature(Tm) of hair fastener shape nucleic acid oligomer probe, promptly at hair fastener shape nucleic acid oligomer probe not with any nucleic acid oligomer sequence hybridization the melting temperature(Tm) of the state of Individual existence at the detecting area and the residing hybridization state of single-point mispairing sample of hair fastener shape nucleic acid oligomer probe, it is the melting temperature(Tm)-5 ℃ of single-point mismatch hybridization state, to the detecting area of hair fastener shape nucleic acid oligomer probe with mate sample hybridization state of living in fully, promptly mate fully between the scope of melting temperature(Tm)+5 ℃ of hybridization state.
The hair fastener shape nucleic acid oligomer probe of gene chip of the present invention is unmarked probe.Chip base surface coverage one is to the electrical isolation and the electrochemistry inert material membrane of some molecular layers.Hair fastener shape nucleic acid oligomer probe is chemically bound in the chip base surface.
The method of the above-mentioned gene chip with hairpin-shaped probes of preparation of the present invention, its step comprises
1, design, synthetic nucleic acid oligomer probe,
Nucleic acid oligomer probe after design is synthetic has following structure: exists one section or plurality of sections by the nucleic acid oligomer sequence that the gene order of detected sample determines, be called detecting area; Exist one section or plurality of sections nucleic acid oligomer sequence and with the nucleic acid oligomer sequence that this sequence is complementary, be called the cane district; Nucleic acid oligomer sequence in the cane district and form hair fastener shape duplex structure with the nucleic acid oligomer sequence of its coupling;
According to matching way and the base number of sample nucleic acid oligomer sequence in the hybrid experiment according to the nucleic acid in the cane district of following requirement design hairpin-shaped probes: the melting temperature(Tm) of hair fastener shape nucleic acid oligomer probe, in the melting temperature(Tm)-5 of single-point mismatch hybridization state ℃ between the scope of the melting temperature(Tm) of mating the hybridization state fully+5 ℃.
2, at least one hair fastener shape nucleic acid oligomer probe is solidificated on the chip base surface.
Described nucleic acid oligomer probe is unmarked nucleic acid oligomer probe.Chip base surface coverage one is to the electrical isolation and the electrochemistry inert material membrane of some molecular layers.The present invention is chemically bound at least one hair fastener shape nucleic acid oligomer probe on the chip base surface.
The method of utilizing the said gene chip to carry out gene test of the present invention, chip base adopts conductive substrates, and its step comprises:
1, substrate is applied control of Electric potentials;
2, regulate the surface potential of substrate, probe under the monitoring different potentials and the crossover process that is detected sample;
3, determine that according to monitoring result this gene chip is detected the best hybridization conditions of sample to such;
4, under top condition, detect such and be detected sample, realize that this gene chip is detected sample to such and mates identification with the nucleic acid oligomer sequence of single-point mispairing fully.
Advantage of the present invention and positively effect:
A) utilize chemical reaction that unmarked hairpin-shaped probes is cured to electrode, unmarked hairpin-shaped probes being evenly distributed on electrode surface, the chip of different batches preparation repeats functional, and same chip can use repeatedly.Can significantly reduce the average cost that detects each time like this.The problem of the cost costliness that exists in the traditional die technology, poor repeatability, poor stability improves.Unmarked hairpin-shaped probes of the present invention and sample all need not carry out any mark, and chip can reusable characteristics in addition, make and use cost all can reduce greatly.
B) utilize the present invention to obtain to the identification of the nucleic acid oligomer sequence of coupling and single-point mispairing fully
Fig. 1 shows the chip surface and the nucleic acid oligomer sequence sample hybridization (square number strong point) of coupling fully that is connected to unmarked hairpin-shaped probes respectively, be connected to the chip surface of unmarked hairpin-shaped probes and the nucleic acid oligomer sequence sample hybridization (triangle number strong point) of single-point mispairing, the electrode surface (diamond data points) that does not have any nucleic acid oligomer sequence of bonding chain, and bonding unmarked hairpin-shaped probes chip surface but do not carry out Mott-Schottky measuring result under any nucleic acid oligomer hybridization (circular data point) four kinds of situations.The result shows, has when being connected to the nucleic acid oligomer sequence hybridization of the chip surface of unmarked hairpin-shaped probes and coupling fully with the unmarked hairpin-shaped probes that has been connected to the chip surface of unmarked hairpin-shaped probes and the nucleic acid oligomer sequence hybridization of single-point mispairing, the electrode surface that does not have any nucleic acid oligomer sequence of bonding chain, bonding but do not carry out other three kinds of information slips that any nucleic acid oligomer hybridizes to reveal diverse character.Pay special attention to, the result of back three kinds of situations coincides together fully, shows that the present invention has identification and mates ability with the nucleic acid oligomer sequence of single-point mispairing fully.The present invention realizes that the principle of discerning is: satisfying under the situation of design requirements of the present invention, about the numerical value of the free energy (aforementioned three kinds of melting temperature(Tm)s are relevant with these free energys) of molecule suitable, make that the probe and the bonding state of nucleic acid oligomer sequence of coupling fully are stable (therefore can form heteroduplex), and with the bonding state of the nucleic acid oligomer sequence of single-point mispairing be unsettled (therefore can not form heteroduplex).
C) utilize the present invention, can be at above-mentioned B) the basis on, further chip surface character is regulated by surface potential, obtain the top condition of the nucleic acid oligomer recognition sequence of coupling and single-point mispairing fully.Because after current potential is applied to electrode, the free energy of the nucleic acid oligomer molecule of the various forms on surface can change with the difference of current potential, causes the change of corresponding melting temperature(Tm) thus.The nucleic acid oligomer free energy of different shape is with the velocity of variation difference of potential variation, so the change difference of melting temperature(Tm).Be adjusted to the melting temperature(Tm) of hair fastener shape nucleic acid oligomer probe like this through control of Electric potentials, in the melting temperature(Tm) of single-point mismatch hybridization state with mate optimum value between the melting temperature(Tm) of hybridization state fully, thereby obtain the best identified of the nucleic acid oligomer sequence of coupling and single-point mispairing fully.
Fig. 2 shows the result of real-time detection crossover process.The result shows, by design to unmarked hairpin-shaped probes, select again suitable surface potential (among the figure for-0.7V), just can reach the identification of under identical hybridization conditions chip to the crossover process of the coupling fully of nucleic acid oligomer sequence and single-point mispairing, that is: chip has response to the sample of nucleic acid oligomer sequence of coupling fully, and the response that the sample of single-point mispairing not can observe.Fig. 2 display chip simultaneously can reuse.
Description of drawings:
The character comparison diagram of the Mott-Schottky mapping of the different chip surfaces of Fig. 1
Diamond data points comes from the chip surface (I) that has connect one deck 12 carbon carboxylic acid membrane on silicon base, the circular data point comes from the chip surface (II) that has connect unmarked hairpin-shaped probes on above-mentioned surface (I), the square number strong point comes from the chip surface (IV) that is after the chip surface (III) behind the nucleic acid oligomer sequence hybridization of above-mentioned surface (II) and coupling fully, triangle number strong point come from the nucleic acid oligomer sequence hybridization of above-mentioned surface (II) and single-point mispairing.This figure shows that the chip surface (III) of coupling has and the different character in other surface [(I), (II), (IV)] fully.The result of surface (I), (II), (IV) overlaps, and result and other surface on surface (III) are completely different.
The same gene chip that is connected to unmarked hairpin-shaped probes of Fig. 2 carries out the Real Time Observation synoptic diagram of hybrid experiment repeatedly under different potentials
The nucleic acid oligomer sequence sample that added the single-point mispairing in the time of 400 seconds added the nucleic acid oligomer sequence sample of coupling fully in the time of 700 seconds.When current potential is selected when suitable, only the nucleic acid oligomer sequence sample of coupling has response to chip to adding fully.This figure display chip simultaneously can reuse.
Fig. 3 ac impedance measurement pond synoptic diagram
1--working electrode 2--metal 3--channel of nitrogen 4--chip
5--reference electrode 6--counter electrode 7--tetrafluoroethylene electrochemical cell
The chip that has wherein connect unmarked hairpin-shaped probes constitutes working electrode, and saturated calomel electrode is as reference electrode, and platinum is as counter electrode.
Fig. 4 gene chip with hairpin-shaped probes structural representation
11--conductor or semiconductor-based end 12--electric insulation layer 13--cane 14--detecting area
Embodiment:
Prepare the p-silicon face that covers with carboxyl terminated individual layer organic membrane according to existing method [seeing document T.Strother, et al., J.Am.Chem.Soc.122:1205 (2000)].Selecting one section nucleic acid oligomer sequence of human body cancer suppressor gene p53 is sample.Studies show that in p53, have a plurality of sites all may be because of the simple point mutation cause cancer takes place.Here one section detected normal gene sequence of Xuan Zeing be (representing) 5 with 219 '-GGC ATG AAC CGG AGG-3 '; the carcinogenic sequence of simple point mutation be (representing) 5 with 220 '-GGCATG AACTGG AGG-3 ', wherein the base of underscore is arranged is the base of position of undergoing mutation for black matrix and italic.The base sequence of unmarked hair fastener type probe of design is 5 '-
GCGAGCCCT CCG GTT CATGCC
GCTCGC-(CH
2)
6-NH
2-3 ', wherein the base of underscore is the cane district, the base sequence of detecting area and normal gene sequence are mated fully.The melting temperature(Tm) that calculates is: T
m(single-point mismatch hybridization)=58 ℃, T
m(unmarked hairpin-shaped probes)=67 ℃, T
m(coupling hybridization fully)=66 ℃.Utilize unmarked hairpin-shaped probes-NH
2The peptide bond that condensation reaction between the carboxyl of group and organic membrane forms is cured to [10mM EDAC/10mM NHS (MES buffer on the silicon face organic membrane with unmarked hairpin-shaped probes, pH=6.5) activation, 4 ℃ of fixation reactions 4 hours], make the chip that is connected to unmarked hairpin-shaped probes.Can discern fully the gene fragment of coupling and single-point mispairing in two ways:
First kind, under different electropotentials, measure the character of Mott-Schottky mapping.Sample and chip hybridization [the 20mM Tris-HCl+100mM MgCl of sample at first
2, pH=8.1 buffered soln drips at chip surface, and 37 ℃ were reacted 6 hours down].After chip cleans [2 * SSPE, 37 ℃, ultrapure water then], with 20mM Tris-HCl, 100mM MgCl
2, pH=8.1 solution is supporting electrolyte, utilizes impedance detection pond as shown in Figure 3, under the deoxygenation situation, utilize electrochemical workstation (commodity, CHI 660A, Shanghai) carry out the measurement of different electropotentials (V) lower surface electric capacity (Cs) under the 5mV voltage of alternating current between the 45kHz at 15kHz, with (C
s)
-2To the V mapping, get Mott-Schottky figure.Do a chip liquid simultaneously and hybridize the contrast experiment of buffered soln merely.Recognition result as shown in Figure 1.The Mott-Schottky figure that mates sample fully is different with the contrast experiment's, and the Mott-Schottky of single-point mispairing sample schemes and the overlapping of contrast experiment.
Second kind, Real Time Observation crossover process under a certain optimum electrode current potential.Utilize impedance detection pond shown in Figure 3, utilize electrochemical workstation (commodity, CHI 660A, Shanghai), under the deoxygenation situation, at 20mMTris-HCl+100mM MgCl
2, pH=8.1 buffered soln drips sample, direct viewing crossover process down.The known coupling fully of usefulness and the experiment of the single-point mispairing standardized solution optimum electrode current potential that finds identification at first for this reason.Be in the present embodiment-0.7V.(0.7V),, detect surface capacitance (C at 25kHz 5mV voltage of alternating current and optimum electrode current potential along with the adding of sample
s).When adding the sample of single-point mispairing, electric capacity does not have transition, when adding the sample that mates fully, and electric capacity generation transition.The result is as first width of cloth figure among Fig. 2 (0.7V).
Claims (5)
1. a gene chip with hairpin-shaped probes comprises the silicon substrate, and chip base surface coverage one is to the electrical isolation and the electrochemistry inert material membrane of some molecular layers; With the oligonucleotide probe that is chemically bound on this material membrane, have at least an oligonucleotide probe to comprise detecting area, there is one section or plurality of sections oligonucleotide sequence by the gene order decision of detected sample, it is characterized in that this oligonucleotide probe also comprises the cane district, the oligonucleotide sequence that has one section or plurality of sections oligonucleotide sequence and be complementary with this sequence, oligonucleotide sequence in the cane district and have duplex structure with the oligonucleotide sequence of its coupling and detecting area constitutes hair fastener shape structure together; The matching way of the nucleic acid in cane district and base number satisfy following condition:
The melting temperature(Tm) of hair fastener shape oligonucleotide probe is in the melting temperature(Tm)-5 of single-point mismatch hybridization state ℃, between the scope of the melting temperature(Tm) of mating the hybridization state fully+5 ℃.
2. gene chip with hairpin-shaped probes as claimed in claim 1 is characterized in that described hair fastener shape oligonucleotide probe is unmarked probe.
3. method for preparing gene chip with hairpin-shaped probes as claimed in claim 1, its step comprises
1) design, synthetic oligonucleotide probe,
Oligonucleotide probe after design is synthetic has at least one to have following structure: exists one section or plurality of sections by the oligonucleotide sequence that the gene order of detected sample determines, be called detecting area; Exist one section or plurality of sections oligonucleotide sequence and with the oligonucleotide sequence that this sequence is complementary, be called the cane district; Oligonucleotide sequence in the cane district and have duplex structure with the oligonucleotide sequence of its coupling and detecting area constitutes hair fastener shape structure together;
According to detected sample oligonucleotide sequence, matching way and base number according to the nucleic acid in the cane district of following requirement design hairpin-shaped probes: the melting temperature(Tm) of hair fastener shape oligonucleotide probe, in the melting temperature(Tm)-5 of single-point mismatch hybridization state ℃ between the scope of the melting temperature(Tm) of mating the hybridization state fully+5 ℃;
2) at least one hair fastener shape oligonucleotide probe is chemically bound in cover the silicon substrate surface one on the electrical isolation and electrochemistry inert material membrane of some molecular layers.
4. the method for preparing gene chip with hairpin-shaped probes as claimed in claim 3 is characterized in that described hair fastener shape oligonucleotide probe is unmarked oligonucleotide probe.
5. method of utilizing the described gene chip with hairpin-shaped probes of claim 1 to carry out gene test, chip base adopts the silicon substrate of conduction, and its step comprises:
1) substrate applies control of Electric potentials to silicon;
2) surface potential of adjusting chip base, hair fastener shape oligonucleotide probe under the monitoring different potentials and the crossover process that is detected sample;
3) determine that according to monitoring result this gene chip is detected the best hybridization conditions of sample to such;
4) under top condition, detect such and be detected sample, realize that this gene chip is detected sample to such and mates identification with the oligonucleotide sequence of single-point mispairing fully.
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