CN2570290Y - AIDS virus original position nucleic acid chip - Google Patents

Abstract

The utility model discloses an AIDS virus original position nucleic acid chip, which relates to a diagnostic and screening device for AIDS. The AIDS virus original position nucleic acid chip comprises a chip, a microelectrode array fixed on a substrate and electrode leads fixedly connected with the microelectrodes. Every two microelectrodes in the microelectrode array form a sensor. Different AIDS virus nucleic acid fragments are respectively and correspondingly solidified as probes in the zone which is composed of microelectrodes and from which each sensor generates signals, and the probes form an AIDS virus probe array. The utility model can detect many fragments of various AIDS virus nucleic acids simultaneously with high efficiency, high speed and automatic convenience, and without marking so as to realize the diagnosis of AIDS and the tracking of the principle thereof. The utility model has the advantages of low cost of chips, accuracy, sensitiveness and short detecting time, and can be used for the quantitative analysis of AIDS viruses.

Description

AIDS virus in-situ nucleic acid chip

Technical field:

The utility model relates to the diagnosis and the screening apparatus of acquired immune deficiency syndrome (AIDS), be specially adapted to virus of AIDS fast, detection by quantitative.

Background technology:

Along with acquired immune deficiency syndrome (AIDS) spreads in the whole world, virus of AIDS HIV the infected is increasing, up to the present, acquired immune deficiency syndrome (AIDS) is not still had effective prophylactic treatment measure, and therefore, it is particularly important to set up sensitive special, simple and rapid virus of AIDS detection technique.

Biochip technology has important application prospects with its efficient height, the characteristics that contain much information in virus of AIDS detects.The currently reported biochip technology that relates to the virus of AIDS diagnosis mainly contains two classes.

One class is that gene to be measured is carried out sequential analysis.As application on January 19th, the 1999 laid-open U.S. Patents US Patent5 of U.S. Affymetrix company, 861,242 " Array of nucleic acidprobes on biological chips for diagnosis of HIV and methods of usingthe same ", disclose a kind of high density oligonucleotide probe array chip that is made of up to ten thousand oligonucleotide of the HIV of being used for sequential analysis, each Nucleotide to the HIV conserved sequence in this probe array has at least four complementary oligonucleotide probes with it.Probe adopts the photoconduction chemical synthesis, generates to be fixed on the solid support thing.

Another kind of be characterizing gene fragment with pathogenic agent to be measured as probe, with extract in the blood sample, hybridize through the DNA or the RNA of amplification label fluorescence, judge yin and yang attribute.As Wuhan University's application State Intellectual Property Office's invention disclosed patent on October 25th, 2000 " integrated diagnosis chip of venereal disease and preparation method thereof ", notification number CN1271098, this disclosure of the Invention a kind of integrated diagnosis chip and this chip production method of venereal disease, it is to be carved with micro-pit array at glass or silicon chip glazing, these arrays are divided into eight zones, adopt common chemical combination bonding method to be fixed with characterizing gene or its primer of the tool diagnostic significance that comprises eight kinds of venereal disease cause of diseases such as AIDS successively in this cheats slightly.Shanghai Bodao Gene Technology Co. Ltd. application State Intellectual Property Office in August 29 calendar year 2001 the invention disclosed patent " be used for infectious disease hepatitis B virus; treponema pallidum; the gene chip of HIV (human immunodeficiency virus) and hepatitis C virus diagnosis ", notification number 1310236, this disclosure of the Invention a kind of infectious diseases HBV that is used for, TP, the gene chip of HIV and HCV diagnosis, this gene chip comprises monitoring and two systems of medical diagnosis on disease of detecting, wherein disease diagnosing system is provided with the probe of the conserved regions sequence that is selected from above-mentioned four kinds of infectious diseases pathogen nucleic acids, can detect above-mentioned four kinds of infectious virus simultaneously.

Above-mentioned existing chip technology, at first on probe constituted, the former adopted high density arrays to carry out sequential analysis to target gene, but makes chip cost too high, is unsuitable for daily clinical diagnosis; The latter only adopts a kind of characterizing gene fragment, and the somatotype that can not carry out virus of AIDS detects.

Secondly, chip detection is to utilize the fluorescent marker method of labels targets gene such as fluorescein, and the fluorescent signal that is incorporated into target gene on the chip with the acquisition of high resolution fluorescent scanning instrument carries out analytical test.There are some serious deficiencies in fluorescent marker method, as detect need before the labels targets gene, wash-out just can not detect behind the binding label to need to finish also at hybridization, test set complexity and cost height, testing conditions there is extremely strict requirement, operate miscellaneously, be difficult to realize automatization, miniaturization or the like.

Once more, can only whether there be the qualitative detection of virus of AIDS, and can not carries out the quantitative analysis of virus of AIDS.

The problems referred to above that existing virus of AIDS chip technology exists have seriously limited it and have applied.

Summary of the invention:

The purpose of this utility model is the problems referred to above that exist at existing virus of AIDS biochip technology, provide a kind of with low cost, need not mark, easy to use, fast efficient, the AIDS virus in-situ nucleic acid chip that can carry out somatotype and quantitative analysis simultaneously to virus of AIDS.

AIDS virus in-situ nucleic acid chip, comprise substrate, be fixed on on-chip micro-electrode array, each microelectrode is connected with testing circuit by contact conductor respectively, in micro-electrode array, constitute a transmitter with two microelectrodes, space length between two microelectrodes is 10 μ m-10mm, space length between the transmitter is wanted more than the 10 μ m at least, be solidified with different AIDS virus nucleic acid fragments respectively accordingly as probe in the zone of each transmitter generation signal that micro-electrode array constituted, form the virus of AIDS probe array.

The utility model adopts and make the identical microsensor array of structure on substrate, the feature nucleic acid fragment that solidifies a kind of virus of AIDS on each transmitter is as probe, constitutes in use to realize its purpose by the AIDS virus in-situ nucleic acid chip that the response original position of transmitter is obtained probe and target nucleic acid reaction information.

Above-mentioned substrate is silicon or quartzy or semi-conductor or insulating material such as glass or plastics.

Above-mentioned microelectrode is gold or silver or metallic conduction materials such as copper or aluminium.

Above-mentioned AIDS virus nucleic acid probe is the characterizing gene fragment of virus of AIDS, and the corresponding a kind of virus of AIDS of a kind of probe comprises the virus of AIDS of various hypotypes; One probe be solidificated on the surface of one or two electrode of a transmitter be solidificated on the transmitter two interelectrode substrates or be solidificated in a transmitter two microelectrodes and between the zone on.

Above-mentioned various virus of AIDS characterizing gene fragments are selected from the conserved regions of various HIV viruses, and various HIV virus conserved regions target genes have designed 34 kinds of probes altogether for existing public technology, and its sequence is as shown in table 1:

Table 1 AIDS virus nucleic acid probe sequence

The probe numbering	HIV target gene code	The HIV target gene	Probe sequence (5 '-3 ')
				1	SK29	501-518	ACTAGGGAACCCACTGCT
2	SK30	589-605	GGTCTGAGGGATCTCTA
				3	SK31	552-585	ACCAGAGTCACACAACAGACGGGCACACACTACT
4	SK89	9432-9449	AGGAGCTGGTGGGGAACG
				5	SK90	9577-9596	GTGCTGGTGAGAGTCTAGCA
6	SK91	9524-9561	TTGAGCCCTGGGAGGTTCTCTCCAGCACTAGCAGGTAG
				7	SA38	1551-1578	ATAATCCACCTATCCCAGTAGGAGAAAT
8	SK39	1638-1665	TTTGGTCCTTGTCTTATGTCCAGAATGC
				9	SK19	1595-1635	ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC

10	SK145	1336-1395， 1150-1121	AGTGGGGGGACATCAAGCAGCCATGCAAAT
				11	SK101	1506-1482， 1258-1234	GCTATGTCAGTTCCCCTTGGTTCTC
12	SK150	1507-1480， 1259-1232	TGCTATGTCACTTCCCCTTGGTTCTCTC
				13	SK102	1403-1435， 1158-1190	GAGACCATCAATGAGGAAGCTGCAGAATGGGAT
14	SK100	1377-1395， 1132-1150	ATCAAGCAGCCATGCAAAT
				15	SK104	1646-1667， 1401-1422	CCTTTGGTCCTTGTCTTATGTC
16	SK109	1351-1391	AGATAGGATTGCAGAAGTGTGTCAGGATGTACAACCGACC
				17	P1	764-768	TACATCAGGCCATATCACCTAC
18	P2	1041-1066	TGAAGGGTACTAGTACTTCCTGC
				19		993-1027	AGGGCCTATTGCACCAGGCCAGATGAGAGAACC
20	SK68	7801-7820	AGCAGCAGGAAGCACTATGG
				21	SK69	7922-7942	CCAGACTGTGAGTTGCAACAG
22	SK70	7841-7875	ACGGTACAGGCCAGACAATTATTGTCTGGTATAGT
				23	SK122	6569-6589	CAAAGCCTAAAGCCATGTGTA
24	SK123	6870-6891	TAATGTATGGGAATTGGCTCAA
				25	SK129	6586-6611	TGTAAAATTAACCCCACTCTGTGTTA
26	CO1	7855-7874	ACAATTATTGTCTGGTATAG
				27	CO2	7970-7989	AGGTATCTTTCCACAGCCAG
28	CO3	7895-7934	TGAGTTGCAACAGATGCTGTTGCGCCTCAATAGCCCTCAG
				29	POL	2356-2381	TGGGAAGTTCAATTAGGAATACCAC
30	P3	2637-2663	CCTACTATACAAATCATCCATGTATTC
				31	P4	2508-2545	ATGAGACACCAGGGATTAGATATCAGTACAATGTGCT
32		4085-4103	ATTAGCAGGAAGATGGCC

33	P5	4207-4225	TACTCCTTGACTTTGGGG
				34	P6	4313-4171	CCACCAACAGGCGGCCTTAACCGCAGCACTGGTGAAATT

The detected object that is applicable to AIDS virus in-situ nucleic acid chip of the present utility model is the target material, can be the DNA (thymus nucleic acid) or the RNA (Yeast Nucleic Acid) of various virus of AIDS, probe can be the sequence synthetic oligonucleotide according to the feature nucleic acid fragment of virus of AIDS.Since on transmitter to a kind of probe should be arranged, and on the array chip that constitutes by a plurality of transmitters to the probe groups that is made of multiple probe should be arranged, therefore can detect comprising all known various virus of AIDS.

When the sample solution that will contain the target material contacts with this original position chip, the target material is caught by corresponding probe, the interaction of probe-target will cause the variation of transmitter response (electrical parameter), thus, can be incorporated into the action attitude to target material-probe specific on each transmitter in the original position chip by electric parameter measurement and monitor in real time, thereby realization is to the mensuration of target material.

The utility model and existing biochip relatively have following obvious advantage and unusual effect.

The utility model one is to adopt microelectronics to make chip, can be mass-produced, the 2nd, adopt the characterizing gene section of various HIV viruses to make up the nucleic acid probe array of virus of AIDS, can detect the virus of AIDS of various hypotypes all-sidedly and accurately, and need not highdensity probe array, therefore greatly reduce chip cost.

The utility model adopts microsensor, variation by a kind of electrical signal due to measuring probe-target combination, original position, the directly interaction of detection probes-target in real time, the one, the HIV target nucleic acid need not the coupling fluorescein etc. marker, easy to use, the 2nd, required electronic metering equipment is simple, is easy to realize miniaturization, automatization, and is also not high to the requirement of environment for use.Efficient fast, accurately sensitive, easy to detect, can detect a plurality of fragments of multiple hiv virus nucleic acid simultaneously, realize the dynamic tracking of diagnosis, the state of an illness and the curative effect of acquired immune deficiency syndrome (AIDS) etc.

When AIDS virus in-situ nucleic acid chip of the present utility model uses, the one, do not exist the not cleaning of binding label, the 2nd, need not mark and reduced the sample preparation time, the 3rd, as long as have enough binding capacities to make electrical signal that considerable change be arranged and need not wait question response to finish, therefore can shorten detection time greatly.

The utility model with utilize sensor signal variable quantity and transmitter on the binding capacity of target viral nucleic acid relevant, can be used for HIV virus is carried out quantitative analysis.

Description of drawings:

Fig. 1 is the structural representation of a kind of AIDS virus in-situ nucleic acid chip of the present utility model.

Among Fig. 1,1 is substrate, and 2 is microelectrode, and 3 is contact conductor, and 4 is substrate region between transmitter two electrodes, and probe is not shown in Fig. 1.

Specific embodiments:

A kind of AIDS virus in-situ nucleic acid chip of the present utility model, as shown in Figure 1.Constitute by substrate 1, microelectrode 2 arrays and contact conductor 3 thereof, immobilization probe array.It specifically is constructed as follows:

1) above-mentioned substrate 1 adopts insulation or semiconductor material and usual method to make plain film shape, and the material of use can be inorganicss such as silicon single crystal, glass, quartz, corundum sheet, also can be organism such as polystyrene, pi, as shown in Figure 1, the long 20mm of substrate, wide 13mm.

2) on substrate, make micro-electrode array with microelectronics:

(1) evaporation or sputtering electrode metallic substance on substrate 1 as gold or silver or platinum or nichrome, are made the metallic film of thick 100nm-5000nm.

(2) adopt photolithography to make electrode pattern, adopt the reactive ion etching method again, make the array and the lead-in wire thereof of microelectrode 2 by the electrode pattern corrosion.Two rows on substrate, have been adorned to parallel alignment, the array that every row is made up of 6 transmitters, the long 0.2-2mm of each microelectrode, wide 0.2-2mm, the space length of two microelectrodes of each transmitter is 0.2-2mm, the wide about 0.1-1mm that goes between is solidified with AIDS virus nucleic acid probes different in 12 respectively on the substrate between two microelectrodes of 12 transmitters.

3) at the sensor surface stationary probe:

AIDS virus nucleic acid probe of the present invention has designed 34 kinds, shown in preceding table 1 altogether according to the design of HIV characterizing gene fragment.Can all select for use, also can select wherein several as required for use, as on chip shown in Figure 1, selecting 1-12 probe in the table 1 for use.

Probe stationary also can be fixed on the sensor electrical interpolar substrate region on the pair of electrodes of transmitter.When probe stationary during in gold, niobium oxides, iridium oxide, platinum, titanium, tantalum, tungsten and other metallic surface, these metallic surfaces can by with probe on organic sulfydryl be connected to form stable conjugates.As 1 ' or 3 ' end mark on the synthesized dna probe of sulfydryl can form stable conjugates with metal such as gold.When probe stationary as the glass surface of substrate the time, can be by making its epoxy group(ing) functionalization with the epoxy silane reaction, at epoxy group(ing) and 5 ' on glass-amino-derivatized oligonucleotide probe reaction, form secondary amine covalency connection, and probe is connected to the surface of glass.Derivative as 5 ' aldehyde or carboxylic acid, amino and phosphoric acid can combine with the polystyrene that hydrazides, diazotization activation and nitrogen base are modified respectively.

The fixing of probe can adopt micropipet or mini sprinkler or syringe needle that various probe pointwises are distributed on each corresponding site of substrate surface behind synthetic various probes.

Use AIDS virus nucleic acid original position chip of the present invention, the sensing detection that AIDS virus nucleic acid is detected can be with sophisticated various electricity or electromagnetism method, below wherein alternating-current inducing defecation by enema and suppository is made a presentation:

Conductance for alternating current G _AC=ε A/d, in the formula, A is that useful area, the d of electrode is interelectrode operating range, ratio between two for chip used be a constant; And ε is a specific inductivity, depends on electrode surface and the character and the state of medium therebetween.In testing process, G _ACTo change along with the variation of the amount that whether has nucleic acid hybridization, nucleic acid hybridization on the transmitter.Therefore can original position be carried out in the reaction of probe and target nucleic acid detect in real time, thereby judge whether to exist certain virus of AIDS, and can determine the amount of this AIDS virus nucleic acid by the conductance for alternating current of measured chip upper sensor.

Claims (5)

1, a kind of AIDS virus in-situ nucleic acid chip comprises substrate, is fixed on on-chip micro-electrode array, and each microelectrode is connected with testing circuit by contact conductor respectively; It is characterized in that per two microelectrodes constitute a transmitter, space length between two microelectrodes is 10 μ m-10mm, space length between the transmitter is wanted more than the 10 μ m at least, be solidified with different AIDS virus nucleic acid fragments respectively accordingly as probe in the zone of each transmitter generation signal that micro-electrode array constituted, form the virus of AIDS probe array.

2, AIDS virus in-situ nucleic acid chip according to claim 1, the long 20mm of substrate that it is characterized in that this chip, wide 13mm, two rows on substrate, have been adorned to parallel alignment, the array that every row is made up of 6 transmitters, each microelectrode is long to be 0.2-2mm, wide is 0.2-2mm, thick is the metallic film of 100nm-5000nm, the space length of two microelectrodes of each transmitter is 0.2-2mm, the wide 0.1-1mm of contact conductor is solidified with AIDS virus nucleic acid probes different in 12 respectively on the substrate between two microelectrodes of 12 transmitters.

3, AIDS virus in-situ nucleic acid chip according to claim 1 is characterized in that each AIDS virus nucleic acid probe is solidificated on the surface of one or two microelectrode of a transmitter.

4, AIDS virus in-situ nucleic acid chip according to claim 1 is characterized in that each AIDS virus nucleic acid probe is solidificated on the substrate between transmitter two microelectrodes.

5, AIDS virus in-situ nucleic acid chip according to claim 1, it is characterized in that each AIDS virus nucleic acid probe be solidificated in a transmitter two microelectrodes and between the zone on.

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