CN1769491A - False complementary peptide nucleic acid probe biochip and detection method based on SPR principle - Google Patents
False complementary peptide nucleic acid probe biochip and detection method based on SPR principle Download PDFInfo
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Abstract
This invention discloses a complementary peptidenucleic acid probe biological chip, wherein the solid-phase holder of the said chip joins with the complementary peptidenucleic acid probe. This invention also relates to the chip checking method basing on the plasma resonance SPR principle, which includes the crossing of the sample and the checking probes; clearing and wiping off the uncrossed samples; making the polarised light checking on each probe cross zone that is on the checking biological chip by using the plasma resonance equipment; using plasma resonance SPR principle to analyze the complementary peptidenucleic acid probe crossing reaction to obtain the result. This invention uses the pcPNAs as the cross probe, compraring to the ordinary DNA probe, it has higher sensitivity and especial charater, which not only avoids PCR reaction, but also delows the sample treatment request, simples the operation steps and shortens the checking time.
Description
Technical Field
The invention relates to the field of biochips, in particular to a biochip taking pseudo-complementary peptide nucleic acid as a probe and a detection method based on the surface plasmon resonance principle.
Technical Field
With the wide application of molecular biology in the medical field, the chip technology will be an indispensable new content in clinical disease diagnosis in the future. The chip technology is generated along with the human genome project, is an important progress of molecular biology and medical diagnosis technology in recent years, has the outstanding characteristics of high speed, high flux, high parallelism, diversification, miniaturization, automation and the like, becomes a hotspot of the current biomedical technology research, and shows great development potential and application value in a plurality of fields of gene diagnosis, drug development, toxicity analysis, pathogen detection and the like.
At present, although the chip technology has been developed and attracted attention by people, there are still many problems in several aspects of sample preparation, probe synthesis and fixation, molecular labeling, data reading and analysis, etc., and the price is high, thereby severely restricting the wide application and further development of the chip technology. In particular, in the detection of gene chips, due to the limitations of labeling techniques and hybridization signal amplification techniques, it is common practice to perform appropriately programmed amplification of samples before labeling and analysis, most commonly PCR, in order to improve detection sensitivity and specificity. The PCR amplification requires both the design of primers and the construction of PCR instruments, and requires different amplification systems, amplification methods and amplification conditions for the target sequences of the detection objects, thereby greatly increasing the workload and the working difficulty.
Peptide Nucleic Acid (PNA) is a DNA mimic designed by computer assistance, and like DNA, is polymerized from monomers of four bases ATCG, except that the backbone structures of the two are different: DNA is a pentose phosphate backbone formed by phosphodiester linkages; PNAs are composed of repeating N-2-aminoethylneuraminic acid units linked by amide bonds. PNA is not easily degraded by protease, nuclease, etc. and does not bind to protein in serum, but can bind to DNA by complementary pairing principle of ATCG four bases. Because the pseudopeptide skeleton of PNA is neutral, PNA has stronger affinity because of no electrostatic repulsion in the hybridization between DNA or RNA, and the stability and specificity of the combination are greatly improved. The PNA is introduced into the field of detection chips as a detection probe, so that the sensitivity and specificity of gene detection of the biochip are greatly improved, and the PNA becomes a hotspot for designing and synthesizing the current detection chip probe. Although Peptide Nucleic Acid (PNA) shows good hybridization properties to single-stranded DNA and RNA, it can only be polypurine or polypyrimidine PNA when hybridized with double-stranded DNA, and has too strict sequence restriction, which greatly limits the detection capability of PNA probe for double-stranded DNA, especially the operation of complete genome, crude sample or unpurified sample can not meet the requirement of molecular biology. Therefore, for the PNA probe to be used for DNA detection, the processing of the sample necessarily includes the processes of extraction, purification, denaturation, etc. of DNA.
Pseudocomplementary peptide nucleic acids (pcPNAs), which are base-modified derivatives of peptide nucleic acids, have recently been developed in the study of DNA double strand invasion hybridization properties of peptide nucleic acids. That is, two PNA hybridization fragments against a sequence of double-stranded DNA (dsDNA) are designed using the sequence as a target fragment. And adenine (A) in the PNA fragment is entirely substituted with diaminopurine (D) and thymine (T) is entirely substituted with mercaptouracil: (sU) is substituted. Thus, when two of the groups contain D,spcP of UAs NAs react with the dsDNA, pcPNAs bind to the corresponding target sequence on both strands of dsDNA simultaneously, forming stable PNA-dsDNA-PNA complexes. However, pcPNAs themselves are interchain by D andssteric hindrance of the U-steric structure prevents binding, which ensures that pcPNAs do not bind complementarily to themselves while recognizing the target sequence. pcPNAs-double strandsThe specific recognition pattern of DNA significantly extends the possible target sequence composition of DNA. In fact, any mixed base site on dsDNA except for sites with too high GC content (A + T. ltoreq.40%) can be used as target sequence for pcPNA. At present, no report exists on the gene or protein detection aspect by using pcPNAs as probes.
Although there are no reports of using pcPNAs as probes for gene or protein detection, we have determined that pcPNAs may theoretically be more advantageous as nucleic acid hybridization probes than conventional PNAs. Since conventional PNA can be hybridized with single strand or dsDNA, but only poly-homologous pyrimidine PNA (triple strand Invasion) or poly-homologous purine PNA (double strand Invasion) due to the strong binding force between the PNA strands when bound to dsDNA in strand Invasion mode, this clearly reduces the prevalence and selectivity of PNA as probe. pcPNAs can be designed into any base sequence complementary to the target sequence without poly-homologous purines or poly-homologous pyrimidines; and pcPNAs can bind to corresponding target sequences in both strands of dsDNA simultaneously to form stable PNA-dsDNA-PNA complexes. The dsDNA capable of being hybridized and combined with pcPNAs can even be supercoiled compact plasmid DNA, so that the detection capability of the DNA can be greatly improved, the method is particularly suitable for application research of complete genome, crude samples or unpurified sample operation, the complexity of sample treatment is reduced, and the preparation time and the operation process of the early-stage sample are simplified, thereby providing possibility for clinical rapid gene detection, particularly rapid gene detection under field conditions. Compared with PNA probes, the pcPNAs probe applied to gene detection reduces the purification and denaturation processes of the sample, omits the denaturation treatment time in hybridization, and simplifies the control of the hybridization temperature condition.
Based on the above theory, the inventors have conducted a pioneering study of the binding activity of pcPNAs to DNA and protein, and the probe characteristics and hybridization characteristics of the pcPNAs as a chip detection probe, and compared them with the conventional chip detection probes. On the basis, the pcPNAs chip is introduced into the surface plasma resonance sensor for the first time, and the specificity and the sensitivity of the chip serving as a detection probe, the method and the characteristics of practical detection application and the like are studied in detail.
Surface Plasmon Resonance (SPR) sensors are novel optical sensor devices that have been rapidly developed in recent years, and are characterized in that real-time (real time) and online (on line) detection of interactions between biomolecules is possible, and labeling and purification are not required, and therefore, the SPR sensor is one of the research hotspots in the current international sensor field. The basic principle is that after incident light is coupled and excited by a group of optical devices, usually a high-refractive-index prism, sequentially placing a coupling layer (mostly a metal film) with a low refractive index at a certain angle, surface plasmon resonance occurs on the interface of the metal film, that is, the incident light interacts with free electrons on the interface surface, wherein part of incident light waves are absorbed by charge density waves, and then the incident light cannot be totally reflected, so that the intensity of the reflected light is greatly reduced, the reflection angle is obviously deviated, and the change of the intensity and the angle of the reflected light has a certain proportional relation with the number of biological substance molecules adsorbed on the interface.
The Self-assembly (SAM) technique of thiol compound surface monolayer immobilized as a probe was reported since the beginning of the 20 th century 80 years, and rapidly received attention from all parties because it satisfied several major requirements as a model interface, including firm binding to the substrate, controlled molecular orientation ordering, controlled external surface properties, and controlled thickness. In general, SAMs are formed based on the strong chemical bonding of a thiol compound to a substrate material (e.g., gold, silver, platinum, etc.). For example, on the surface of the gold film, a mercapto group is oxidized to form a sulfur alloyed bond. The chemical reaction formula is as follows: . The chemical bonding force between Au-S is strong, the bonding energy is as high as 184k J/mol, and few other groups can compete with the bonding force, so that the selectivity of the combination is ensured. Meanwhile, the SAM is arranged closely and orderly and has stronger resistance to acid, alkali, ion penetration and the like. The method for fixing the probes can ensure that the probes are firmly combined, orderly arranged and uniformly distributed. Based on the strong chemical combination of sulfydryl and metal surface, the present invention combines sulfydryl modified pcPNAs probe with metal film of surface plasma resonance equipment to produce chip of pcPNAs probe array for detecting various target genes and proteins.
Disclosure of Invention
One of the objects of the present invention is: providing a detection biochip using pseudo-complementary peptide nucleic acid as a probe; the invention also aims to provide a method for detecting the biochip taking the pseudo-complementary peptide nucleic acid as the probe based on the surface plasmon resonance principle. By the method, the sample can be amplified by bypassing, the sample processing step is simplified, and the sensitivity and specificity of detection are improved; and the optical signal is taken as the detection signal, so that the method is stable and easy to detect, and the equipment and the technology for detecting and analyzing the result are greatly simplified. Therefore, the characteristics of high flux, high sensitivity and high specificity of the biochip technology can be utilized to overcome the main defects of the existing detection chip.
One of the technical solutions adopted to achieve the above objects of the present invention is a biochip for detection using pseudo-complementary peptide nucleic acids as probes, characterized in that: and connecting a pseudo-complementary peptide nucleic acid detection probe on the chip solid phase carrier.
The pseudo-complementary peptide nucleic acid probe is connected with a nano metal film which is attached to a chip solid phase carrier, has surface plasma resonance response characteristics and has the thickness of 10nm-150 nm.
The second technical scheme adopted by the object of the invention is as follows: a method for detecting a biochip using pseudo-complementary peptide nucleic acid as a probe based on the surface plasmon resonance principle is characterized in that: the method comprises the following steps:
spreading a nano metal film (with the thickness of 10nm-150 nm) with surface plasma resonance inductivity on the surface of a chip solid phase carrier; connecting a pcPNAs probe preparation chip on the surface of the nano metal film;
(II) processing the sample, and carrying out hybridization reaction with the probe on the biochip;
and thirdly, carrying out polarized light detection on each probe hybridization area by using surface plasma resonance equipment, and analyzing the array probe hybridization reaction by using the surface plasma resonance principle to obtain a result.
The method for paving the nanogold film with surface plasma resonance inductivity on the surface of the chip solid phase carrier in the step (I) comprises the following steps:
(1) preparing a nano gold solution by a trisodium citrate reduction method (Frens method);
(2) paving a surface plasma resonance nano metal film with uniform property by taking N- β - (aminoethyl) -gamma aminopropyl trivalent oxysilane (APTMS) as a core;
(3) and drying the paved nano metal film by nitrogen and sealing for later use.
The method for connecting the pcPNAs probe array on the surface of the nano gold film in the step (I) comprises the following steps:
(1) cleaning the prepared nano gold film;
(2) connecting the pcPNAs probe array by a surface self-assembly monolayer technology to generate a self-assembly array of probes;
(3) and cleaning the chip, removing the unassembled probes, drying and sealing for later use.
The method for processing a specimen in the second step includes:
(1) extracting DNA or RNA of bacteria, cells and tissues; nucleic acid substances such as DNA, cDNA generated by PCR or RT-PCR reactions;
(2) or extracting proteins from bacteria, cells and tissues, and secreting proteins from fluids.
The step (II) of carrying out hybridization reaction between the specimen and the probe on the gene chip comprises the following steps:
(1) loading the sample on a detection biochip taking the pseudo-complementary peptide nucleic acid as a probe;
(2) injecting the processed specimen into a micro-flow reaction tank for hybridization reaction; carrying out hybridization reaction on the specimen and the detection probe under the conditions of:
a) for the DNA chip, the buffer was 20mM sodium phosphate buffer (pH7.0) and the reaction volume was
10-50 mul, the flow rate of the circular reaction is 5-20 mul/min, the reaction temperature is 0-99 ℃, and the reaction time is 5 minutes-50 hours;
b) for the protein chip, the buffer solution is a protein chip hybridization buffer solution, and the buffer solution consists of 20mM HEPES, 1mM DTT, 0.1mM EDTA, 50mM KCl, 5% glycerol and 200 mug/ml bovine serum albumin, the reaction volume is 10 mug to 50 mug, the flow rate of the circular reaction is 5 to 20 mug/min, the reaction temperature is 0 to 99 ℃, and the reaction time is 5 minutes to 50 hours;
(3) and washing to remove the non-hybridized and combined specimen.
The surface plasmon resonance detection in the step (three) includes:
(1) performing surface plasma resonance scanning on the chip hybridization reaction area, namely performing polarized light detection on each probe hybridization area on the biochip by using surface plasma resonance equipment;
(2) and analyzing the scanning result to obtain a chip detection result.
The invention has the following advantages and effects:
(1) the invention adopts pcPNAs as hybridization probes, and has higher sensitivity and specificity compared with common DNA probes;
(2) the invention adopts pcPNAs as hybridization probes, compared with DNA probes or PNA probes, not only PCR reaction is avoided, but also the processing requirement of samples is reduced, the operation steps are simplified, and the detection time is shortened;
(3) the invention adopts pcPNAs as hybridization probes and uses the surface plasma resonance principle to detect signals, does not need expensive detection instruments, and reduces the use cost and experimental conditions of the chip.
The term "biochip" used in the present invention is an array constructed by attaching biological macromolecules or tissues to chip solid phase carriers such as glass, ceramics, metal sheets or nylon membranes, nitrocellulose membranes, etc. Examples of biochips include gene (nucleic acid) chips, cell chips, protein chips, antibody chips, or tissue chips, and the like.
The nucleic acid of the invention includes DNA, RNA, cDNA or peptide nucleic acid, pseudo-complementary peptide nucleic acid.
The term "pseudo-complementary peptide nucleic acid" as used herein is a base-modified derivative of peptide nucleic acid, i.e., two PNA probes are designed with a sequence on both strands of dsDNA as the target fragment. In the probe, all of adenine (A) is substituted with diaminopurine (D) and all of thymine (T) is substituted with mercaptouracil (A), (B), (C), (sU) is substituted.
The protein of the invention comprises various antibodies, antigens, nucleic acid function-related enzymes and the like.
The term "nano metal film" used in the present invention refers to a metal film layer having a thickness of 10nm to 150nm formed of nano metal particles having a diameter of 10nm to 150nm, and may be a gold film, a silver film, a platinum film, etc. The preferred nano-metal film layer used in the present invention is a nano-gold film with a thickness of 50 nm.
In a word, the successful development of the technology can really realize the characteristics of high flux, specificity, sensitivity and rapidness of the chip technology. The kit can quickly and accurately detect and identify pathogenic microorganisms, can also research one or more specific genes or expression products related to the specific genes, can research the relation between the genes and proteins and diseases, verify the genes related to the diseases, develop and screen new medicaments, perform molecular diagnosis of the diseases, track and prognosis of treatment processes and the like, and provides important guidance for prevention and treatment of clinical diseases; meanwhile, the technology can also be applied to the aspects of investigation of battlefield pathogen distribution, early diagnosis of war injury infection, early discovery of biological warfare agents and the like performed by primary inspectors under the field condition, and can generate better economic benefit and social benefit.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a schematic diagram of the basic structure of a probe pcPNAs designed in accordance with the present invention;
FIG. 2 is a schematic diagram of the basic structure of the surface plasmon resonance sensor of the present invention;
FIG. 3 is a schematic diagram of a surface plasmon resonance chip system according to the present invention.
Referring to FIG. 1, the pcPNAs probe designed by the present invention has a structure pattern with a pseudopeptide backbone as its core, and its base mainly comprises D,SU, G, C are provided. In the figure, a represents D andssteric hindrance between U significantly affects the binding between PNA complementary monomers; but can combine with common DNA copies to form stable pairs; b indicates that the composition of the basic backbone of DNA is different from that of PNA, DNA is a nucleic acid sugar-phosphate backbone, and PNA is a pseudopeptide backbone.
Referring to fig. 2, the surface plasmon resonance sensor apparatus used in the detection method of the present invention is configured to include a: a prism; b: a chip; c: incident polarized light; d: reflecting the light; e: an optical signal collector; f: a signal amplifier; g: a digital signal converter; h: and (4) a computer.
Referring to fig. 3, the surface plasmon resonance chip of the present invention comprises a chip solid phase carrier 4, a nano metal film 5, and a probe 6; in the figure, 7 is a reaction cell for hybridization detection, 8 is an optical signal collector of a surface plasmon resonance sensor device, 1 is a laser beam, 2 is a polarizing film, and 3 is a prism.
Detailed Description
Example 1 a method for spreading a 50nm thick nanogold film with surface plasmon resonance inductivity on a glass slide comprises the following steps:
(1) preparing gold sol by a trisodium citrate reduction method (Frens method): heating and boiling 100ml of 0.01% chloroauric acid aqueous solution, adding 0.75ml of 1% trisodium citrate aqueous solution under stirring, allowing the gold chloroauric acid aqueous solution to turn into mauve within 2 minutes, boiling for 15 minutes, cooling, and recovering to original volume with distilled water.
(2) And paving a gold film:
A. cleaning the glass slide by acid washing, putting the glass slide into N- β - (aminoethyl) -gamma-aminopropyl trivalent oxylsilane (APTMS)/toluene solution for refluxing for 12 hours, cleaning, taking out, drying by nitrogen, and placing the glass slide in a clean and dry place for later use;
B. and soaking the treated glass slide in a nano gold solution for 12 hours, then soaking the glass slide in a 0.01mol/L PDDA aqueous solution for 10-20min, taking out and cleaning the glass slide with ultrapure water. After drying, the glass slide is moved into hydroxylamine/chloroauric acid solution and soaked for 0.5 hour, and a bright gold film layer is formed on the surface of the glass slide.
Example 2 a method for preparing pcPNAs probe array on gold film surface by using surface self-assembly monolayer technology, comprising the following steps:
(1) the prepared nano gold film is coated in piranha solution (30% H)2O2H in different concentration2SO41: 3), then repeatedly washing with double distilled water, and drying with nitrogen;
(2) dripping SH-pcPNAs/PBS buffer solution with 500nM concentration to react with the nano gold film in a self-assembly way, and reacting for 24 hours at 100% humidity to generate a self-assembly array of the probe;
(3) and respectively sucking dry buffer solution, sealing with 6-mercaptohexanol, cleaning with double distilled water, and drying with nitrogen.
Example 3 one-step method for rapid treatment of specimens by the reagent Chelex-100 to extract bacterial DNA, the steps are as follows:
(1) collecting a pure culture colony ring by using a sterile inoculating ring, and placing the pure culture colony ring in a 1.5ml sterilized Eppendorf tube;
(2) adding 50 mu l of 5% Chelex suspension, mixing uniformly, adding 20mg/ml proteinase K, digesting in 56 ℃ water bath for more than 2h, then in boiling water bath for 7.5min, immediately placing in ice bath for 3min, centrifuging at 12000r/min for 5min, and taking supernatant as DNA sample.
Example 4 cell lysate lysates of cells, crude extracts of nuclear proteins, the steps are:
(1) shearing the tissue into small pieces, adding 200-300 mul of homogenate into each 20mg of tissue, and homogenizing at a low temperature of 4 ℃;
(2) standing for 10min, adding 90 μ l 10% NP-40, shaking vigorously for 30s, centrifuging at 4 deg.C for 15min at 800g, and discarding the supernatant;
(3) dissolving the precipitate in 5ml of lysis solution, incubating for 30min at 4 ℃ with shaking, centrifuging for 15min at 4 ℃ at 13000g, and taking the supernatant as a nucleoprotein extract;
example 5 hybridization reaction of DNA samples with the pcPNAs probes on the GeneChip, the procedure was:
(1) assembling the prepared chip on the surface of the manufactured surface plasma resonance sensor prism, injecting 20 mu l of 20mM sodium phosphate buffer solution (PH7.0) into a flow reaction tank, preheating (45 ℃) for 30 minutes, and reading the initial value of the reflection angle of the reflected light;
(2) injecting the processed sample supernatant into a 20-mul flow reaction tank, circularly flowing and hybridizing for 3 hours at the temperature of 45 ℃, and reading the reflection angle numerical value of the reflected light of each probe sampling point;
(3) the flow reaction cell was filled with 20. mu.l of 20mM sodium phosphate buffer (pH7.0), and the chip was washed at 37 ℃ for 1 hour, and repeated 3 times.
Example 6 hybridization of protein samples with pcPNAs probes on protein chips, the procedure was:
(1) assembling the prepared chip on the surface of the manufactured surface plasma resonance sensor prism, injecting 20 mu l of 10mM TE buffersolution into a flow reaction tank, preheating (37 ℃) for 30 minutes, and reading the initial value of the reflection angle of the reflected light at the moment;
(2) injecting the processed protein sample supernatant into a 20-mul flow reaction tank, hybridizing for 2 hours at 37 ℃, and reading the reflection angle value of the reflected light of each probe sampling point;
(3) the flow reaction cell was filled with 20. mu.l of 10mM TE buffer, and the chip was washed at 37 ℃ for 1 hour, and repeated 3 times.
Example 7 DNA chip detection procedure using pcPNAs probe was:
gonococci were provided by the bacterial laboratory of the clinical laboratory in the university Hospital, and the standard of identification was in accordance with "national clinical laboratory practice".
Chelex-100 was purchased from BioRad. A5% Chelex buffer was prepared containing 1% NP-40, 1% Tween-20, 0.03% SDS.
Gonococcal pcPNAs probes: HS- (CH)2)6-5’-sU CGCsUsU CsU CGGsU CGCsU-3’；5’-DGCGDCCGDGDDGCGD-(CH2)6-SH-3', synthesized by Applied Biosystems, usa;
TCEP HCL: tis (2-carboxyethenyl) -Phospholine Hydrochloride, Pierce, USA, 0.287mg TCEP dissolved in 1000ul water to prepare 1mmol/L TCEP;
(1) collecting a ring of pure cultured gonococcus colonies by using a sterile inoculating loop, and placing the ring in a 1.5ml sterilized Eppendorf tube;
(2) adding 50 mu l of 5% Chelex suspension, mixing uniformly, adding 2 mu l of proteinase K (20mg/ml), digesting in a water bath at 56 ℃ for 6h, then putting in the ice bath for 3min immediately after passing through a boiling water bath for 7.5min, centrifuging at 12000r/min for 5min, and taking the supernatant as a DNA sample.
(3) The prepared nano gold film is coated on piranha (30% H)2O2H in different concentration2SO41: 3), then repeatedly washing with double distilled water, and drying with nitrogen.
(4) 25 μ M SH-pcPNAs was added with an equal volume of 1mmol/L TCEP, reduced at 37 ℃ for 30 minutes, and PBS buffer (1.44g NaH) was added2PO4、8g NaCL、0.2g KCL、0.24g KH2PO4Adding 800mL of deionized water for dissolution, adjusting the pH value to 6.5 by using HCL, performing constant dissolution to 1L, and performing autoclaving), wherein the final concentration of SH-pcPNAs is 500 nM;
(5) and dropwise adding SH-pcPNAs/PBS buffer solution with 500nM concentration to perform self-assembly reaction on the nano gold film, and reacting for 24 hours at 100% humidity to generate the self-assembly array of the probe.
(6) Assembling the prepared chip on the surface of the manufactured surface plasma resonance sensor prism, injecting 20mM sodium phosphate buffer (PH7.0)20 mul (10 mul/min) into a flow reaction tank, preheating (45 ℃) for 30 minutes, and reading the initial value of the reflection angle of the reflected light at the moment;
(7) injecting the processed sample supernatant into a 20 mul flow reaction tank (5 mul/min), circularly hybridizing for 3 hours at 45 ℃, and simultaneously reading the reflection angle value of the reflected light of each probe point on a surface plasma resonance sensor device;
(8) the flow reaction cell was filled with 20. mu.l of 20mM sodium phosphate buffer (pH7.0) (10. mu.l/min), and the chip was washed at 37 ℃ for 1 hour and repeated 3 times.
Example 8 protein chip detection was performed using pcPNAs probe withthe following steps:
design of pcPNAs Probe based on the nucleic acid binding site of NF-. kappa.B (GGGACTTTCC), HS- (CH2)6-5’-AGT TGA GGG GAC TTT CCC AGG C-3’；
Protein chip hybridization buffer: 20mM HEPES, 1mM DTT, 0.1mM EDTA, 50mM KCl, 5% glycerol, 200. mu.g/ml bovine serum albumin;
(1) cutting liver tissue into small pieces, adding homogenate A (mmol/L: Hepes10, Na)3VO4 1、MgCl21.5, KCl 10, NaF 50, EDTA 0.1, EGTA 0.1, phenylmethylsulfonyl fluoride0.5, dithioritol 1 and 0.02% protease inhibitors cocktail, pH7.9)10ml, low temperature homogenization at 4 ℃;
(2) standing for 10min, adding 90 μ l 10% NP-40, shaking vigorously for 30s, centrifuging at 4 deg.C for 15min at 800g, and discarding the supernatant;
(3) the precipitate was redissolved in lysate B (mmol/L: Hepes 20, NaCl 420, MgCl)21.5, EDTA 1, EGTA 1, dithiothreitol 1, phenylmethylsulfonyl fluoride0.5, glycerol 20% and 0.02% protease inhibitors cocktails, pH7.9) 5ml, incubating with shaking at 4 ℃ for 30min, centrifuging at 4 ℃ for 15min at 13000g, and taking the supernatant as a nucleoprotein extract;
(4) the prepared nano gold film is coated on piranha (30% H)2O2H in different concentration2SO41: 3) soaking and cleaning in the solution, and then usingAnd (5) repeatedly cleaning with double distilled water and drying with nitrogen.
(5) 25 μ M SH-pcPNAs was added with an equal volume of 1mmol/L TCEP, reduced at 37 ℃ for 30 minutes, and PBS buffer (1.44g NaH) was added2PO4、8g NaCL、0.2g KCL、0.24g KH2PO4Adding 800mL of deionized water for dissolution, adjusting the pH value to 6.5 by using HCL, performing constant dissolution to 1L, and performing autoclaving), wherein the final concentration of SH-pcPNAs is 500 nM;
(6) and dropwise adding SH-pcPNAs/PBS buffer solution with 500nM concentration to perform self-assembly reaction on the nano gold film, and reacting for 24 hours at 100% humidity to generate the self-assembly array of the probe.
(7) Assembling the prepared chip on the surface of the manufactured surface plasma resonance sensor prism, injecting 20 mul (10 mul/min) of protein chip hybridization buffer solution into a flow reaction tank, standing for 30 minutes at 0 ℃, and reading the initial value of the reflection angle of the reflected light;
(8) injecting 20 mul of the processed sample supernatant into a 20 mul flow reaction tank (5 mul/min), carrying out circular hybridization for 3 hours at 25 ℃, and reading the reflection angle value of the reflected light of each probe sampling point;
(9) the flow reaction cell was filled with 20. mu.l of protein chip hybridization buffer (10. mu.l/min), and the chip was washed at 0 ℃ for 1 hour and repeated 3 times.
Example 9 hybridization detection and analysis of results
Experimental detection adopts a refraction light angle modulation mode, and the angle modulation range is from 40 degrees to 70 degrees; the angular resolution precision is higher than 0.001 degree; detecting the minimum concentration of less than 1 × 10-11And M. According to the detection target DNA/protein of the designed chip probe, the corresponding angle response change values are respectively measured by utilizing the concentration gradient of the standard substance to draw a target DNA/protein concentration/angle response curve. And after the experiment is finished, comparing the angle change value of the experiment result with the corresponding position of the curve to detect the existence and concentration of the target DNA/protein.
Claims (5)
1. A detection biochip using pseudo-complementary peptide nucleic acid as a probe is characterized in that: and connecting a pseudo-complementary peptide nucleic acid detection probe on the chip solid phase carrier.
2. The detection biochip according to claim 1, wherein: the pseudo-complementary peptide nucleic acid probe is connected with a nano metal film which is attached to a chip solid phase carrier, has surface plasma resonance response characteristics and has the thickness of 10nm-150 nm.
3. The detection biochip according to claim 2, wherein: the nano metal film with surface plasma resonance response characteristics attached to the chip solid phase carrier can be a gold film, a silver film or a platinum film.
4. The detection biochip according to claim 2, wherein: the false complementary peptide nucleic acid detection probe is connected to the nano metal film by adopting a probe curing-self-assembly monolayer technology.
5. A method for detecting the biochip according to claim 2 based on the principle of surface plasmon resonance, comprising: the method comprises the following steps:
(1) loading the sample on a detection biochip using the pseudo-complementary peptide nucleic acid as a probe according to claim 2;
(2) and carrying out hybridization reaction on the sample and the detection probe under the conditions of:
a) for the DNA chip, the buffer solution is 20mMsodium phosphate buffer solution (PH7.0), the reaction volume is 10-50 mul, the flow rate of the circular reaction is 5-20 mul/min, the reaction temperature is 0-99 ℃, and the reaction time is 5 minutes-50 hours;
b) for the protein chip, the buffer solution is a protein chip hybridization buffer solution, the buffer solution consists of 20mM HEPES, 1mM DTT, 0.1mM EDTA, 50mM KCl, 5% glycerol and 200 mug/ml bovine serum albumin, the reaction volume is 10 mug to 50 mug, the flow rate of the circular reaction is 5 to 20 mug/min, the reaction temperature is 0 to 99 ℃, and the reaction time is 5 minutes to 50 hours;
(3) washing to remove the specimen which is not hybridized and combined;
(4) and carrying out polarized light detection on each probe hybridization area on the biochip by using surface plasma resonance equipment, and analyzing the pseudo-complementary peptide nucleic acid probe hybridization reaction by using the surface plasma resonance principle to obtain a result.
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| CNB2005100573362A CN100378228C (en) | 2005-10-21 | 2005-10-21 | False complementary peptide nucleic acid probe biochip and detection method based on SPR principle |
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| CNB2005100573362A CN100378228C (en) | 2005-10-21 | 2005-10-21 | False complementary peptide nucleic acid probe biochip and detection method based on SPR principle |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102661932A (en) * | 2012-04-17 | 2012-09-12 | 王利兵 | Method for identifying specific DNA sequences in germplasm resources based on surface plasma resonance technology |
| CN105648070A (en) * | 2016-02-25 | 2016-06-08 | 青岛科技大学 | Method for detecting nucleic acid or cells based on enzymatic cycle amplification and nano-particle reinforced SPR (surface plasmon resonance) |
| CN106323918A (en) * | 2016-05-31 | 2017-01-11 | 中国科学院苏州纳米技术与纳米仿生研究所 | Local plasma optical imaging ellipsometry biosensor and preparation method thereof |
| CN111595916A (en) * | 2019-09-18 | 2020-08-28 | 南京农业大学 | NF-kB electrochemical detection method based on screen printing electrode |
-
2005
- 2005-10-21 CN CNB2005100573362A patent/CN100378228C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102661932A (en) * | 2012-04-17 | 2012-09-12 | 王利兵 | Method for identifying specific DNA sequences in germplasm resources based on surface plasma resonance technology |
| CN102661932B (en) * | 2012-04-17 | 2014-07-23 | 王利兵 | Method for identifying specific DNA sequences in germplasm resources based on surface plasma resonance technology |
| CN105648070A (en) * | 2016-02-25 | 2016-06-08 | 青岛科技大学 | Method for detecting nucleic acid or cells based on enzymatic cycle amplification and nano-particle reinforced SPR (surface plasmon resonance) |
| CN106323918A (en) * | 2016-05-31 | 2017-01-11 | 中国科学院苏州纳米技术与纳米仿生研究所 | Local plasma optical imaging ellipsometry biosensor and preparation method thereof |
| CN111595916A (en) * | 2019-09-18 | 2020-08-28 | 南京农业大学 | NF-kB electrochemical detection method based on screen printing electrode |
| CN111595916B (en) * | 2019-09-18 | 2023-10-03 | 南京农业大学 | NF- κB electrochemical detection method based on screen printing electrode |
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| CN100378228C (en) | 2008-04-02 |
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