CN201372286Y - Rapid high-flux foodborne microorganism testing instrument - Google Patents

Abstract

The utility model discloses a rapid high-flux foodborne microorganism testing instrument, which comprises a testing instrument main frame. The rapid high flux foodborne microorganism testing instrument is characterized in that a reaction tank is arranged on the testing instrument main frame, the reaction tank is provided with a peptide nucleic acid probe biochip with portions immersed into the reaction tank, a prism is assembled on the peptide nucleic acid probe biochip, a polarizer is mounted above one end of the prism, a laser generator is arranged above the polarizer, a tester is mounted above the other end of the prism, the surface of the peptide nucleic acid probe biochip is attached with a metal film, and a peptide nucleic acid probe array is manufactured on the surface of the metal film. Successful research of the utility model can really lead characteristics of high flux, specificity, sensitivity, rapidity and the like of the chip technique to be realized. The rapid high flux foodborne microorganism testing instrument can not only rapidly detect and determine foodborne microorganism, but research one or more specific gene or relative expression products, further can research relation of gene, protein and diseases, test relative gene of diseases, develop and screen novel medicine, perform molecular diagnosis of diseases, trace treating process, apply prognosis and the like, provides important guidance effect to prevention and treatment of clinical diseases, and simultaneously, the technique can also be applied for junior inspectors to implement investigation of pathogen distribution on battlefield, early diagnosis of battle injury infection, early detection of biological warfare agent and the like on conditions of field operations, and has fine economic and social benefits.

Description

High-throughput food source property microbial rapid detection instrument

Technical field

The utility model relates to detector, especially relates to high-throughput food source property microbial rapid detection instrument.

Background technology

Along with the widespread use of molecular biology at medical field, chip technology will become an indispensable fresh content in the following clinical disease diagnosis.Chip technology is followed the Human Genome Project and is produced, it is the impressive progress of molecular biology and medical diagnostic techniqu in recent years, its outstanding feature is at a high speed, high-throughput, high parallelism and variation, microminiaturization, automatization etc., become the focus of current biomedical technology research, demonstrated huge development potentiality and using value in a plurality of fields such as gene diagnosis, drug development and oxicity analysis, cause of disease detections.

At present,, obtain attracting attention of common people, still exist many problems, and cost an arm and a leg, thereby seriously restricted the widespread use of chip technology and further develop although chip technology has been obtained significant progress.As on marking method, fluorescent method is maximum method of using at present, but its sensitivity not high, need lucifuge, want rapid detection after hybridization is finished in order to avoid fluorescent quenching, the result detects in addition needs with the price specific installations such as laser scanner of costliness extremely; Isotope-labelling method has the inconvenience of use owing to need isotropic substance and radioautograph, complicated operation, the shortcoming of growing, pollution being arranged consuming time.Especially in the detection of gene chip, by can influencing pcr amplification efficient with fluorescent mark, isotopic labeling commonly used as ³²P and ³³The P transformation period is too short again, all be difficult for promoting the use of, and pcr amplification both needed to design primer, again the PCR instrument, need set up different amplification systems, amplification method and amplification condition to the target sequence of each detected object, this has obviously increased workload and work difficulty greatly.In a word, chip technology the preparation of sample, probe is synthetic with fix, the reading and analyze etc. and still exist many insoluble problems aspect the several of mark of molecule, data.Particularly lower, the poor repeatability of technical costs costliness, complexity, detection sensitivity, analysis are general encloses problem such as narrower and has limited the application of this technology in biological detection.

Peptide nucleic acid(PNA) (Peptide Nucleic Acid, PNA) be a kind of dna analog by computer aided design (CAD), the same with DNA also is that monomer polymerization by four kinds of bases of ATCG forms, just both skeleton structure differences: DNA is the phosphopentose skeleton that is formed by connecting by phosphodiester bond; And PNA is made of the multiple N-2-aminoethyl glycosides propylhomoserin unit that amido linkage connects.PNA is difficult for by degradeds such as proteolytic enzyme, nucleases, also not with serum in protein binding, but can be by the complementary pairing principle and the DNA hybridization combination of four kinds of bases of ATCG.Because the pseudopeptide backbone of PNA is electroneutral, there is not electrostatic repulsion in the hybridization between PNA and DNA or RNA, thereby has stronger affinity, and its bonded stability and specificity all improve greatly.

Be not used for the report of gene or protein chip context of detection though also have at present with PNA as probe, we think, compare with traditional dna probe, and PNA may have more advantage in theory as dna hybrid probe.Because traditional dna probe can only combine with the DNA hybridization of strand, we know that the PCR product all is double-stranded, therefore before hybridization, must double-stranded PCR product be disintegrated and be the dna fragmentation of strand by denaturation process, and in crossover process, must keep low temperature to influence hybridization efficiency to prevent product D NA renaturation, produce false negative result.But it not only can efficiently hybridize with the DNA hybridization of strand PNA as probe, even can also directly combine three chain cpds that form stable DNA-PNA-DNA with chain invasion and attack pattern with double-stranded DNA, therefore increased detectivity greatly to DNA, and can reduce the complicacy of sample disposal, simplify the setup time and the operating process of sample in early stage, thereby provide possibility for clinical rapid gene detects.

Based on above-mentioned theory, we are probe in a creative way with PNA, to PNA and DNA, combination of proteins activity, and it are systematically studied as the probe characteristics of chip detection and hybridization characteristic etc., and compare with existing chip detection probe; On this basis, and first the PNA chip is introduced surface plasma resonance sensor, to its specificity as the chip of detection probes, susceptibility, and method and characteristic that actual detected is used have been carried out detailed research.

Surface plasma body resonant vibration (Surface Plasmon Resonance, SPR) transmitter is the novel optical sensor device that in recent years develops rapidly, its outstanding feature is (real time), online interaction between the detection of biological molecule (on line) in real time, and do not need mark and purifying, be one of research focus of current international sensor field.

Mercaptan compound surface unimolecular layer self-assembly layer (Self-assembledMonolayer as probe stationary, SAM) technology is since early 1980s is reported, because it has satisfied the several major requirements as the model interface, comprise mortise with substrate, controllable molecular orientation ordered arrangement, therefore aspects such as controllable outer surface properties and controllable thickness have obtained the concern of each side soon.Generally speaking, the SAM strong Chemical bond that is based on sulfhydryl compound and base material (as gold and silver, platinum etc.) forms.As on golden film surface, sulfydryl is oxidized and generate the sulphur aurification and close key.Its chemical equation is as follows: 2Au+2RSH → 2Au-SR+H ₂It is very strong that chemical bond between the Au-S is made a concerted effort, and its bond energy is up to 184kJ/mol, seldom have other groups can with its competition, this has just guaranteed the bonded selectivity.Simultaneously, the arrangement of SAM is closely orderly, and acid, alkali, iontophoretic injection etc. are had stronger resistibility.The adopting said method stationary probe can make probe in conjunction with firmly, and arranges in order, is evenly distributed.We are based on this strong chemically combined characteristic of sulfydryl and metallic surface, PNA probe with sulfydryl modification in the utility model combines with the metallic film of SPR equipment, make the chip of PNA probe array, thereby finish various target genes, proteic detection.

Summary of the invention

The purpose of this utility model is to provide the high-throughput food source property microbial rapid detection instrument of peptide nucleic acid(PNA) (PNA) as probe.By the utility model, can walk around sample is increased, the reduced sample treatment step improves the sensitivity and the specificity that detect; And, both stable with optical signalling as detection signal, be easy to again detect, the equipment and the technology that make the result detect, analyze are greatly simplified.Thereby can make full use of the high-throughput of biochip technology, high responsive, high special characteristic, overcome the main drawback that present detection chip exists.And in order further to improve the efficient of chip detection.We are also on the basis of traditional spr sensor, researched and developed a kind of plating method of simpler, easy enforcement, inexpensive nanoporous metal membrane, voluntarily design compilation corresponding information processing and operation control software, small-sized flowable sample injected system has been assembled in design, and be applicable to the temperature control test pool of chip prehybridization, hybridization and rinsing, thereby the high automation and the intellectuality that this transmitter have been had more be applicable to chip detection.Because this biochip that the utility model research provides and the method for rapid detection has hybridization, test set is simple, consistent being easy to grasped, highly sensitive, good reproducibility, the characteristic of applied range etc., it will become can be widely used in common lab, even the biochip of new generation of field environment.

The purpose of this utility model is achieved in that the utility model comprises the detector body frame, it is characterized in that: the pond responds on the detector body frame, some is immersed in the peptide nucleic acid probe biochip of reaction tank on reaction tank, on peptide nucleic acid probe biochip, be assembled with prism, above a prismatical end, polaroid is installed, laser generator installation and measuring device above the prismatical the other end is being arranged above the polaroid; The peptide nucleic acid probe biochip surface attachment metallic film arranged, the metallic film surface point is shaped on the peptide nucleic acid probe array; Metallic film preferably thickness is the nano-gold film of 10nm～150nm.The utility model principle of work be when incident light with certain angle through one group of optics---be generally order on the prism of a high refractive index and place after coupling layer (the mostly being metallic film) coupling of one deck low-refraction excites, surface plasma body resonant vibration takes place on the metallic membrane interface, the unbound electron that is incident light and surface, boundary can interact, wherein the part incident light wave is absorbed by charge density wave, then total reflection can not take place in incident beam, thereby cause catoptrical intensity to weaken greatly, tangible deviation also takes place in reflection angle, and the biological substance molecular amounts of adsorbing on the variation of this catoptrical intensity and angle and the interface how much become certain proportionlity.

The employed term of the utility model " biochip " is to make up the array that forms by biomacromolecule or tissue apposition on glass, pottery, tinsel or substrate materials such as nylon membrane, nitrocellulose filter.The example of biochip comprises gene (nucleic acid) chip, cell chip, protein chip, antibody chip or organization chip etc.

Nucleic acid described in the utility model comprises DNA, RNA, cDNA or PNA.

The employed term of the utility model " peptide nucleic acid(PNA) " is a kind of dna analog of nucleic acid, the same with DNA also is that monomer polymerization by four kinds of bases of ATCG forms, just both skeleton structure differences: DNA is the phosphopentose skeleton that is formed by connecting by phosphodiester bond; And PNA is made of the multiple N-2-aminoethyl glycosides propylhomoserin unit that amido linkage connects.

Albumen described in the utility model comprises various antibody, antigen, nucleic acid function relevant enzyme etc.

The employed term of the utility model " nanoporous metal membrane " is meant that the thickness that is formed by the nm gold particles of diameter between 10nm-150nm is the 10nm-150nm golden membranous layer.The preferred nano metal rete that the utility model uses is the nano-gold film of thickness as 50nm.

The advantage and the effect of present technique invention are as follows:

(1) the utility model adopts PNA as hybridization probe, has higher sensitivity and specificity than common DNA or PNA probe;

(2) the utility model adopts PNA as hybridization probe, has not only avoided the PCR reaction, and has reduced the processing requirements of sample, has simplified operation steps, has shortened detection time;

(3) the utility model adopts PNA as hybridization probe, and uses the SPR technology and carry out signal detection, does not need expensive detecting instrument, has reduced the use cost and the experiment condition of chip.

In a word, the high-throughput that really to realize chip technology, specificity, susceptibility and the characteristics such as quick succeeded in developing of the present utility model.Both can carry out rapid and precise detection and evaluation to pathogenic micro-organism, also can carry out the research of a certain or a plurality of specific genes or relative expression product, can also carry out the research of gene and protein and disease relationship, the checking of disease related gene and the exploitation and the screening of novel drugs, the molecular diagnosis of disease, aspects such as the tracking of therapeutic process and prognosis are for the prevention and the treatment of clinical disease provides important directive function; This technology also can be applied under field condition and implement the investigation of battlefield pathogen distribution, the early diagnosis that war wound infects, the aspects such as early discovery of biological warfare agent by the reviewer of basic unit simultaneously, will produce favorable economic benefit and social benefit.

Description of drawings

Fig. 1 is the utility model structural representation

Embodiment

The utility model comprises detector body frame 9, pond 8 responds on detector body frame 9, some is immersed in the peptide nucleic acid probe biochip 5 of reaction tank 8 on reaction tank 8, on peptide nucleic acid probe biochip 5, be assembled with prism 3, above an end of prism 3, polaroid 2 is installed, laser generator 1 installation and measuring device 4 above the other end of prism 3 is being arranged above the polaroid 2, the thickness that has of peptide nucleic acid probe biochip 5 surface attachment is the nano-gold film 6 of 50nm, and metallic film 6 surface points are shaped on peptide nucleic acid probe array 7.The utility model principle of work be when incident light that laser generator 1 excites with certain angle after golden film 6 couplings that order on the prism 3 of high refractive index is placed one deck low-refraction excite, surface plasma body resonant vibration takes place on golden film 6 interfaces, the unbound electron that is incident light and surface, boundary can interact, wherein the part incident light wave is absorbed by charge density wave, then total reflection can not take place in incident beam, thereby cause catoptrical intensity to weaken greatly, tangible deviation also takes place in reflection angle, and the biological substance molecular amounts of adsorbing on the variation of this catoptrical intensity and angle and the interface how much become certain proportionlity, after reflected light is injected detector 4, detector 4 detects this catoptrical intensity and angle, and will can detect the biological substance molecular amounts after this changing value input Analytical equipment (as computer).

Claims (3)

1, high-throughput food source property microbial rapid detection instrument, comprise detector body frame (9), it is characterized in that: pond (8) responds on the detector body frame (9), go up some at reaction tank (8) and be immersed in the peptide nucleic acid probe biochip (5) of reaction tank (8), on peptide nucleic acid probe biochip (5), be assembled with prism (3), top at an end of prism (3) is equipped with polaroid (2), and the top installation and measuring device (4) of laser generator (1) at the other end of prism (3) arranged in the top of polaroid (2).

2, high-throughput food source property microbial rapid detection instrument according to claim 1, it is characterized in that: be connected with peptide nucleic acid(PNA) detection probes (7) on the peptide nucleic acid probe biochip (5), peptide nucleic acid probe (7) is attached with metallic film (6).

3, high-throughput food source property microbial rapid detection instrument according to claim 1 and 2, it is characterized in that: metallic film (6) is that thickness is the nano-gold film of 10nm～150nm.

Images