CN100344972C - Virus detection system and method thereof - Google Patents
Virus detection system and method thereof Download PDFInfo
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- CN100344972C CN100344972C CNB2004100327170A CN200410032717A CN100344972C CN 100344972 C CN100344972 C CN 100344972C CN B2004100327170 A CNB2004100327170 A CN B2004100327170A CN 200410032717 A CN200410032717 A CN 200410032717A CN 100344972 C CN100344972 C CN 100344972C
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Abstract
The present invention discloses a virus detection system and a detection method thereof. The virus detection system comprises an atomization system, a detection system and an alarm system, wherein the atomization system is connected with the alarm system through the detection system. The virus detection system and the detection method provided by the present invention can directly detect the gas phase of biological macromolecules through the complete atomization of a saliva sample with SARS virus and a piezoelectric crystal sensor has superpower specificity. The method has the advantages of easiness, high speed, early SARS virus diagnosis and high accuracy.
Description
Technical field
The present invention relates to a kind of viral detection system and a kind of method for detecting virus, particularly a kind of detection system of SARS virus and detection method.
Background technology
SARS (SARS (Severe Acute Respiratory Syndrome) has another name called atypical pneumonia, hereinafter to be referred as SARS) is a kind of very serious infectious disease of 21st century latest find.SARS is with known different by mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella and the common pneumonia that Respirovirus caused.Both compare, and the main route of transmission of SARS is closely to propagate by the spittle, and infectiousness is extremely strong.It has been established that, and SARS virus is a kind of novel coronavirus, and its structure is that its particulate is bigger, about 0.1 to 0.12 micron of diameter by the RNA viruses under the protein coat parcel.The molecular mass of coronavirus virus body is 4 * 10
8D
a, at C
sBuoyant density among the Cl is the 1.23-1.24 gram per centimeter
3Buoyant density in sucrose is the 1.15-1.19 gram per centimeter
3, sedimentation coefficient is 300-500S
20W.The SARS virus body is having Mg
2+Keep stable under the situation about existing relatively, to heat, fatsolvent, nonionic detergent, formaldehyde and oxygenant sensitivity.The coronavirus out-of-shape has coating, and the projection of about 20nm is at interval arranged on the coating, makes virion profile such as corona or crown.The coronavirus particle is medium pantomorphic sphere or ellipse, and substrate is narrow, and end is excellent rod shape.The structural proteins of coronavirus comprise N albumen, M albumen, E albumen, S albumen and HE albumen.
Human early have research to coronavirus, and its history can be traced back to 1933.Baudette etc. have described chicken " asthma ", and nineteen thirty-seven is isolated coronavirus first in the chicken body, and the virus of separation is accredited as infectiousness bronchus viroid.Nineteen sixty-five Tyrrell and Byhve isolate a strain virus, called after B814 virus first at external personnel selection tire nose and tracheae cultural method from common cold patient nose washing lotion.Personnel selection embryo tracheaes such as Melntosh in 1967 are cultivated and are separated to a collection of virus from the common cold philtrum, and its representative strains is OC43.Nineteen sixty-eight June Almeida and Tyrell have carried out morphology research to these viruses, and electron microscope observation finds to have on these viral coatings the spinous process of the similar corona of shape, are coronavirus so propose this viroid of name.ICNV's definite designation in 1975 coronaviridae.
After SARS cause of disease coronavirus was determined, the how tame mechanism in the whole world had begun the research of diagnosis and detection technique.At present sars coronavirus detects and the main direction of research and development of diagnostic techniques has two big classes: based on the immunology detection technology of viral antigen and antibody response and at the nucleic acid detection technique (being also referred to as the molecular Biological Detection technology) of viral RNA.
1, immunology detection technology
At present, the SARS immunology detection mainly is to infect caused antibody response reaction at sars coronavirus.The method of studying at present and carrying out clinical trial mainly contains following several:
(1) ELISA (enzyme linked immunological adsorption reaction): be used for SARS patients serum IgM and IgG TPPA, can detect in 10～21 days about the morbidity back greatly.
(2) IFA (fluorescence immunoassay): be used for SARS patients serum IgM TPPA, can detect in back 10 days about morbidity greatly.This is a kind of reliable assay method, but need measure by means of fluorescent microscope.
Main immunology detection as ELISA and IFA etc., by detecting antibody, can be understood virus infections and disease development process now.Yet all there is limitation in these two kinds of methods as the SARS methods for clinical diagnosis.First kind of method of testing ELISA though can measure antiviral antibody reliably, can only just can detect after clinical symptoms was shown effect 20 days, therefore can not give other people viral communication to prevent the infected as early diagnosis.Second kind of IFA method, this method can be measured antibody in back 10 days exactly in infection, but this method needs fixing virus (or viral antigen), fluorescent microscope and experienced inspection technology personnel.
Recently, professor Li Gang of affiliated hospital of Zhongshan University has found its Changing Pattern through to 21 routine SARS patient's different times serum investigation.They have detected IgM and two kinds of antibody of IgG respectively, find in one week of morbidity, and two kinds of antibody all do not produce.IgM antibody occurs in the time of 10-14 days also peaking very soon in morbidity, and about 1/3 patient still can detect IgM antibody in the time of 60 days, disappears substantially in the time of 90 days.IgG type antibody also can detect in the time of 10-14 days, but titre is lower, peaks in the time of 60 days, still maintained high level in the time of 90 days.Find that simultaneously investigated 20 many cases patients have IgG type antibody all in convalescence.
According to the commodity of above-mentioned SARS detection technique exploitation the detection kit that the Chinese Academy of Sciences and Chinese Military Medical Science Institute develop according to the ELISA method and the detection kit of Beijing Institute of Gene Science, Chinese Academy of Sciences's development are arranged; In addition, CDC (CDC) also set up with the SARS patients serum in associated coronavirus IgM and IgG antibody specificity enzyme-linked immunoassay method.
2, molecular Biological Detection technology
Use molecular Biological Detection technology (as PCR) can detect the inhereditary material RNA of the SARS virus in the various samples (blood, ight soil, respiratory secretions, histotomy).The design of primer (PCR experiment key component) and synthetic method thereof announce in the official website of the World Health Organization (WHO) (WHO), and used by many countries in the world.
The advantage of PCR method is to measure the gene of SARS virus, realizes early diagnosis.But it requires that very strong specificity is arranged between primer and the template, and its sensitivity is not high, a lot of false negatives are arranged among the result, this can not be detected with regard to meaning many actual people that carrying virus---and for being easy to regard to the disease of interpersonal close contact transmission, false-negative testing result is indicating potential safety hazard greatly.
According to PCR method, developed the world's first sars coronavirus detection by quantitative kit cooperatively by the ARTUR company of production firm and the BernhardNocht research centre of professional quantitative PCR kit development.Military Medical Science Institute of PLA has also developed coronavirus fluorescent rna real-time fluorescence RT-RNA detection kit.
Early diagnosis to SARS virus is one of sick most crucial steps of control SARS, but still unobvious so far in this respect progress.Still need explore and be applicable to the early stage quick and easy diagnostic method of virus.
Because SARS virus mainly by droplet transmission, has important Practical significance to direct detection of its gaseous state virus.To the initial stage infect patient SARS to carry that virus directly detects be the difficult point of SARS virus research, also be the key subject of attracting attention in the world.The piezoelectric crystal immunosensor utilizes the specific adsorption of extremely strong affinity between antigen-antibody, is potential in the detection of gaseous state biomacromolecule.
Summary of the invention
One of purpose of the present invention provide a kind of easy fast, can early diagnosis and the high viral detection system of accuracy.
Another object of the present invention provides a kind of method of utilizing above-mentioned viral detection system detection virus.
For achieving the above object, the present invention takes following technical scheme:
A kind of viral detection system comprises atomization system, detection system and warning system; Described atomization system links to each other with described warning system by described detection system.
Described atomization system comprises ultrasonic atomizatio generating means, ultrasonic transduction device and atomizing control circuit; Described detection system comprises mass measurement formula immunosensor and testing circuit device; Described warning system is the single-chip microcomputer warning system; Described single-chip microcomputer warning system comprises difference frequency device circuit, single-chip microcomputer treatment circuit and warning circuit.
Described ultrasonic atomizatio generating means is ultrasonic logic shaker; Described ultrasonic transduction device is the piezoelectric ceramic ultrasonic oscillation chip; Described ultrasonic logic shaker links to each other with described piezoelectric ceramic ultrasonic oscillation chip by the atomizing control circuit; Described mass measurement formula immunosensor is the piezoeletric quartz sensor that scribbles the antibody film, and two electrode inserts on the described testing circuit device; Place described piezoelectric ceramic ultrasonic oscillation chip on the support of the described piezoeletric quartz sensor top that scribbles the antibody film; Described piezoeletric quartz sensor that scribbles the antibody film and described piezoelectric ceramic ultrasonic oscillation chip and be placed in the closed container; The described piezoeletric quartz sensor that scribbles the antibody film links to each other with warning circuit with described difference frequency device circuit, single-chip microcomputer treatment circuit.
Described virus can be coronavirus, is preferably SARS virus.Because antibody engages with the epi-position of antigen by two Fab section, this in conjunction with needing very strong structure specificity, therefore, described piezoeletric quartz sensor only can produce specific adsorption to SARS virus, and is very weak to the absorption of other coronavirus or other viruses.
A kind of method for detecting virus, its step is as follows:
(1) gets the various Virus Samples (blood, ight soil, respiratory secretions or histotomy) that contain,, extract, cultivate, be mixed with solution through handling;
(2) get above-mentioned solution, drop to the ultrasonic atomizatio generating means;
(3) the ultrasonic transduction device transmits energy to described ultrasonic generating means, and the solution on it is carried out ultrasonic aerosolization;
(4) mass measurement formula immunosensor and the testing circuit plate that scribbles the antibody film is installed in the closed container, the virus of aerosolization is attracted on the chip of described mass measurement formula immunosensor;
(5) described mass measurement formula immunosensor links to each other with warning device, is adsorbed virus quantity and acquires a certain degree, and warning device promptly sends alerting signal.
Described ultrasonic atomizatio generating means is ultrasonic logic shaker; Described ultrasonic transduction device is the piezoelectric ceramic ultrasonic oscillation chip; Described ultrasonic logic shaker links to each other with described piezoelectric ceramic ultrasonic oscillation chip by the atomizing control circuit; Described mass measurement formula immunosensor is the piezoeletric quartz sensor that scribbles the antibody film, and two electrode inserts on the described testing circuit device; Place described piezoelectric ceramic ultrasonic oscillation chip on the support of the described piezoeletric quartz sensor top that scribbles the antibody film; Described piezoeletric quartz sensor that scribbles the antibody film and described piezoelectric ceramic ultrasonic oscillation chip and be placed in the closed container; The described piezoeletric quartz sensor that scribbles the antibody film links to each other with warning circuit with described difference frequency device circuit, single-chip microcomputer treatment circuit.
A kind of optimal technical scheme is characterized in that: the described Virus Sample consumption that contains is 0.5-1.5 μ L.
A kind of optimal technical scheme is characterized in that: the electric current that described ultrasonic transduction device is 440mA to described ultrasonic generating means energy delivered; The duration of oscillation of described piezoelectric ceramic ultrasonic oscillation chip is 0.09 second, and vibration is spaced apart 0.2 second, and the number of oscillation is 15 times.
A kind of optimal technical scheme is characterized in that: the described concentration that contains Virus Sample is 0.6-4 μ g/ μ L.
Used piezoeletric quartz sensor is a sessile antibody on the electrode of piezoelectric crystal among the present invention, makes the antibody film.When the antibody film ran into airborne viral antigen, antigen combined the generation immune response and makes the quality increase of piezoelectric crystal cause frequency to change with antibody, and reaches the purpose of detection.The biological power of antigen and antibodies all is intermolecular non-covalent bonding force, as hydrogen bond, Van der Waals force, electrostatic interaction, hydrophobic effect.Thereby piezoeletric quartz sensor can be reused after resolving fully.
Piezoeletric quartz sensor is modal a kind of mass measurement formula immunosensor.Its principle is that quartz wafer has a base frequency when vibrating in oscillatory circuit, when the antigen in the sample or antibody be coated on wafer on antibody or antigen when combining, because the increase of load, the corresponding minimizing of oscillation frequency meeting of wafer, its minimizing value has correlativity with the quality that absorption is got on, and its selectivity is the specific reaction that depends on antigen and antibody.
The present invention will be further described below by the drawings and specific embodiments, but and do not mean that limiting the scope of the invention.
Description of drawings
Fig. 1 is the present invention's virus detection system structural representation;
Fig. 2 is a blank wafer baseline stability linearity curve;
Fig. 3 paraoxon immunosensor baseline stability linearity curve;
Fig. 4 is the response curve of blank wafer to paraoxon;
Fig. 5 is the response curve of albumin A film to paraoxon;
Fig. 6 is the detection curve of paraoxon piezoelectric sensor to paraoxon;
Fig. 7 is a SARS piezoeletric quartz sensor baseline stability linearity curve;
Fig. 8 is the detection curve of albumin A film to SARS virus among the PBS;
Fig. 9 is the detection curve of albumin A+SARS antibody membrane to SARS virus among the PBS;
Figure 10 is the response curve of SARS piezoeletric quartz sensor to the PBS aqueous solution;
Figure 11 is the response curve of SARS piezoeletric quartz sensor to SARS virus among the PBS;
Figure 12 is the working curve of SARS piezoeletric quartz sensor to SARS virus among the PBS;
Figure 13 is the detection curve of SARS piezoeletric quartz sensor to No. 01 viral aqueous solution in Guangzhou;
Figure 14 is the detection curve of SARS piezoeletric quartz sensor to PBS;
Figure 15 is the detection curve of SARS piezoeletric quartz sensor to the A1 influenza virus;
Figure 16 is the detection curve of SARS piezoeletric quartz sensor to PBS;
Figure 17 is the response curve of SARS piezoeletric quartz sensor to saliva;
Figure 18 is the response curve of SARS piezoeletric quartz sensor to virus in the saliva;
Figure 19 is the working curve of SARS piezoeletric quartz sensor to virus in the saliva;
Figure 20 is a different crowd saliva response distribution plan.
Embodiment
As shown in Figure 1, a kind of SARS virus detection system comprises Agilent 53131A type frequency meter 8; The quartzy times gold electrode piezoelectric crystal (external diameter * thickness is 12.5 * 0.2mm, and electrode diameter is 6mm) of the quartz piezoelectric crystal sensor 3--9MHz that Beijing 707 factories make; The two D.C. regulated power supplies 1 of DPS-3015; The Yadu ultrasound wave logic shaker 7 that Yadu Science and Technology Co., Ltd. makes; Titanizing piezoelectric ceramics vibration wafer 4 (diameter 2cm, oscillating current 440mA, duration of oscillation 0.09 second, 0.20 second interval time, the number of oscillation 15 times); Air chamber 5; Glass supporter 6 and testing circuit plate 2 are formed.
The two D.C. regulated power supplies 1 of DPS-3015 link to each other with testing circuit plate 2; A cloche is put in testing circuit plate 2 tops, places a quartz piezoelectric crystal sensor 3 in the cloche and links to each other with testing circuit plate 2; Quartz piezoelectric crystal sensor 3 tops are provided with a glass supporter 6; Put a piezoelectric ceramic ultrasonic oscillation chip 4 on the glass supporter 6; Piezoelectric ceramic ultrasonic oscillation chip 4 is connected with the ultrasonic logic shaker 7 in described Yadu; Testing circuit plate 2 is connected with a computing machine by Agilent5313lA type frequency meter 8.
Biological piezoelectric sensor is a substrate with special piezoelectric quartz crystal, and it is big slightly that this crystal is compared stature with other piezoelectric quartz crystal, and because of the needs of biochemical stability, electrode adopts pure gold what is called " doubly gold " technology, and resonant frequency of a crystal is generally at 9-10MHz.
The circuit of piezoeletric quartz sensor is actually the high-frequency oscillating circuits of a quartz crystal frequency stabilization, and its basic structure is that a quartz crystal and two high-speed cmos double input end NAND gate circuit are formed; Two Sheffer stroke gates are formed high-frequency amplifier circuit, quartz crystal and capacitor C
2After the series connection, form the strong positive feedback of amplifying circuit, thereby produce the stable higher-order of oscillation, export host circuit to after amplifying through a NAND gate circuit buffering, owing to adopt high speed CMOS integrated circuit, starting of oscillation easily, and oscillator signal is highly stable.
After antibody film on the piezoeletric quartz sensor runs into antigen, the frequency of piezoelectric crystal is changed owing to antigen combines with antibody, by frequency meter can be with the change transitions of this frequency digital signal and by the meter record.Agilent 5313lA frequency meter 8 provides RS-232 port, the digital value that provides with character style can be transferred to computing machine by this port.
Viral detection system of the present invention can use piezoeletric quartz sensor that gaseous state antigen is detected in real time: two electrodes that scribble the piezoeletric quartz sensor of antibody film insert on the testing circuit plate, put a piezoelectric ceramic ultrasonic oscillation chip on the glass supporter of piezoeletric quartz sensor top, piezoeletric quartz sensor and sonic oscillation wafer and support place the glass air chamber simultaneously, the glass air chamber is the tubular glass bell jar (volume is 500mL) of Ф 85 * 100mm, with microsyringe fluid sample is placed on the sonic oscillation wafer, build glass bell jar, start ultrasonic logic shaker, sonic oscillation is atomized into aerosol with fluid sample, in air chamber, spread, the immune response meeting takes place and produces selective adsorption in the virus that spreads in the gas on antibody membrane, the piezoelectric crystal quality is increased, cause frequency to reduce, frequency change is by the frequency meter record.By real-time detection software, by computer recording and display graphics.
Experimental example 1
1, paraoxon piezoeletric quartz sensor
1.1 reagent
(1), paraoxon antibody, Military Medical Science Institute of PLA provides;
(2), paraoxon (molecular weight: 275, vapor pressure 1599 * X10
-6Pa/20 ℃, Military Medical Science Institute of PLA provides;
(3), soluble protein A, Sigma company;
(4), polyethyleneimine, Acros company;
(5), the 3-aminopropyltriethoxywerene werene, Acros company;
(6), glutaraldehyde, Beijing chemical reagents corporation.
1.2 paraoxon piezoeletric quartz sensor
1.2.1 electrode pre-service
9MHz gold electrode piezoelectric crystal is used the NaOH of 1.2mol/L respectively, and the HCl of 1.2mol/L soaks 5min, respectively washes secondary with redistilled water, ethanol respectively then, and uses alcohol immersion.Dry under the room temperature.
1.2.2 film forming
A certain amount of a-protein is applied to wafer surface with micro syringe, and room temperature is dried the back and is measured frequency, and the antibody with 0.5 μ L is applied on the wafer then, and room temperature is dried the back measured frequency.
Under the piezoeletric quartz sensor detection system of Fig. 1 and above-mentioned test condition, the blank piezoelectric chip of will not filming places circuit, detect 30 minutes, frequency change is 14Hz only, as shown in Figure 2, this is that electricity by wafer causes noise and the influence of neighbourhood noise to detecting, and illustrate that wafer is subjected to environment and influence circuit less, and is stable better.
The paraoxon piezoeletric quartz sensor that makes is connected into circuit, tests its fundamental frequency stability, as shown in Figure 3, the frequency 40Hz that only descended after 20 minutes, this frequency drop-out value is compared and can be ignored with its response to paraoxon.
Detect the response signal of paraoxon with blank wafer, the result as shown in Figure 4, the frequency 7Hz that only drifts about illustrate that blank wafer to not absorption of paraoxon, does not influence detecting basically in 40 minutes.
Albumin A, polyethyleneimine, 3-aminopropyltriethoxywerene werene are the biological sample immobilized reagents of using always.In order to select immobilized reagent preferably, use these three kinds different membrane matrix sessile antibodies respectively, relatively its immobilization effect.
From to detection of antigens, the testing result of three kinds of matrix with albumin A for well.We select to use a-protein to fix antibody in the experiment below.
The albumin A film is to the detection of paraoxon:
Fig. 5 is the response curve of albumin A film to paraoxon.As seen from the figure, the albumin A film has only 48Hz to the response frequency of paraoxon, illustrates that a glair A is not coated with the film of paraoxon antibody, to detecting basically not influence.
The paraoxon piezoeletric quartz sensor is to the detection of paraoxon:
With ultrasonic ultrasonic delay line memory paraoxon is gasified, the fixing paraoxon antibody generation selective adsorption on paraoxon after the gasification and the sensor makes the quality increase and causes that frequency descends.Fig. 6 be albumin A be membrane matrix fixedly paraoxon antibody is to the testing result of gaseous state paraoxon, as can be seen from Fig. 6, response frequency is slow downtrending because of continuous absorption paraoxon, reaches minimum in the time of 5 minutes, minimum droping frequency is 3978Hz.Compare with the testing result of the wafer that has only the albumin A film, during no paraoxon antibody, sensor does not have response substantially, and behind the sessile antibody, response frequency is greatly improved, and signal is obvious.After response frequency reached minimum, sensor slow was resolved, and frequency is increased gradually.
From above experimental result as can be seen, make crosslinking chemical, the piezoeletric quartz sensor that immobilization paraoxon antibody is made with albumin A, can be easily to being detected by the gaseous state paraoxon after the ultrasonic atomizer atomizing, be swift in response, can in 6 minutes, finish, and reversibility be strong.Description of test, piezoeletric quartz sensor can detect in real time to gaseous state micromolecule antigen.
Experimental example 2
The SARS piezoeletric quartz sensor
1, sample and reagent
(1), saliva sample preparation: after gargling, quiet 15 minutes, get the fresh saliva of 2mL, put on the hydro-extractor with 3000 rev/mins speed centrifugal 15 minutes, it is standby to get supernatant liquor.
(2), SARS virus saliva preparation: the SARS virus freeze-dried powder is made the saliva standard solution, and concentration is respectively 4,3,2,1.5,1,0.5mg/mL;
(3), PBS solution: with 8.0g NaCl, 0.2g KCl, 1.44gNa
2HPO
4, 1.44gK
2HPO
4Be dissolved in the 800mL distilled water, adjust its pH value to 7.4, dilute and be 1.0L, packing, autoclaving, room temperature preservation;
(4), SARS horse serum antibody (1:12800 that tires, the analysis of ELISA method; PBS solution), 37.5mg/mL, Military Medical Science Institute of PLA;
(5), No. 01 SARS inactivation of viruses in Beijing (PBS solution), 4mg/mL, PLA Academy of Medical Sciences;
(6), No. 01 SARS inactivation of viruses in Guangzhou (PBS solution), 1.88mg/mL, PLA Academy of Medical Sciences;
(7), the anti-influenza inactivation of viruses in A1 Shanghai, tiring is 1: 128, the CDC institute of viruses;
(8), soluble protein A, Sigma company;
(9), redistilled water.
2, the preparation of SARS piezoeletric quartz sensor
Get the 9MHZ gold electrode, the NaOH solution with 5mL1.2mol/L soaks 5min earlier, uses the HCI of 5mL1.2mol/L to soak 5min after cleaning again, cleans the back with redistilled water and soak 15min in ethanol, dries stand-by in air.
A certain amount of (concrete volume and concentration are subjected to the influence of required fixing antibody quantity) albumin A is applied to wafer surface with microsyringe, room temperature is dried, the SARS antibody that with 0.5 μ LSARS antibody concentration is 15.00mg/mL again is applied on the albumin A film with microsyringe, and room temperature is dried.
3, the selection of gasification mode
Realize that gas phase condition detects SARS virus down, a crucial and important step is that the SARS virus that will be in the solution is gasified totally into gas, and to the requirement of gasification process be: (1), gasification are wanted fully; (2), speed is fast; (3), amount of samples is few; (4), do not change virus structure, do not damage virus; (5), method is simple.
According to above requirement, natural volatility process and ultrasonic atomizatio method have been compared in test.
The nature volatility process
(1) will contain and not contain the PBS solution of SARS virus, under the same conditions respectively with piezoeletric quartz sensor with placing sealed gas chamber, relatively the two causes the decline of frequency because liquid evaporates into gas naturally.The experimental result explanation, the two frequency change is not seen difference, illustrates that the disease caused by infectious water poison is difficult to evaporate under natural volatilization situation.
(2) will contain and not contain the saliva solution of SARS virus, under the same conditions respectively with piezoeletric quartz sensor with placing sealed gas chamber.Because the volatilization naturally of saliva causes frequency to descend.Experimental result explanation, the two frequency is not seen notable difference, illustrate with the virus in the volatilization method saliva naturally also to be difficult to volatilize.
The ultrasonic atomizatio method
The present invention uses the Yadu sonic oscillation instrument and the diameter of the development of Yadu science and technology limited company as 2cm titanizing PZT piezoelectric ceramic wafer fluid samples such as water, saliva to be atomized, and structure, atomization condition such as sonic oscillation time, oscillating current size, vibration interval, the number of oscillation and the fluid sample consumption that detects air chamber carried out detailed experiments.
The experimental result explanation when the fluid sample consumption is big, can produce the drop splash phenomena during ultrasonic atomizatio, and liquid can not atomize fully, makes measurement result unreliable.When amount of samples was 1 μ L, sample can atomize fully, can not produce splash phenomena when ultrasonic atomizatio, the experiment proved that best ultrasonic atomizatio electric current is 440mA, and duration of oscillation is 0.09 second, and vibration is spaced apart 0.2 second, and the number of oscillation is advisable with 15 times; The structure of detection air chamber is put can get on the sensor with atomizing piece and is tested effect preferably.
The baseline stability linearity curve of SARS piezoeletric quartz sensor:
Experiment test SARS piezoelectric immunosensor baseline stability, as can be seen from Figure 7, after ten minutes, the frequency change of SARS piezoeletric quartz sensor tends towards stability basically, its frequency drift differs 41Hz in 30 minutes.Description of test SARS piezoeletric quartz sensor has good stability.
The SARS piezoeletric quartz sensor is to the detection to SARS virus of the detection of SARS virus among the PBS and albumin A film and albumin A+SARS antibody membrane:
Be the relatively selective reaction of antigen and antibody, studied fixing and not fixedly the albumin A piezoeletric quartz sensor of SARS antibody to the response (concentration is 3mg/mL) of SARS virus among the PBS.From testing result, with the piezoeletric quartz sensor that albumin A is only arranged the SARS virus in the PBS solution is detected, frequency change is 762Hz (as shown in Figure 8), and the frequency change that scribbles the piezoeletric quartz sensor of SARS antibody is 4758Hz (as shown in Figure 9), two figure comparative descriptions albumin As are little to the detection signal influence, mainly are that the antibody on the rete makes the increase of wafer quality and causes that frequency descends with viral the combination.For whether further checking is the association reaction of antibody and antigen, the present inventor has been following one group of contrast experiment again.
The SARS piezoeletric quartz sensor reaches the detection of SARS virus wherein to PBS:
For comparing of the response of SARS piezoeletric quartz sensor to virus, respectively to the PBS aqueous solution with contain viral PBS aqueous solution (concentration is 3mg/mL) and detect, result such as Fig. 9, Figure 10, can find out from two figure comparisons, the SARS piezoelectric immunosensor is 2251Hz to the response of PBS, much smaller than the response (4758Hz) to SARS virus, the decline of this explanation frequency mainly is that antibody combines (2507Hz) that causes with virus, therefore can detect virus.This experiment has illustrated that also the SARS piezoeletric quartz sensor has absorption to water vapor, and the PBS aqueous solution has certain influence to detection.
The influence of SARS antibody concentration:
Table 1 is that the amount of a-protein is certain, scribbles the response of the piezoeletric quartz sensor of 0.5 μ L variable concentrations SARS antibody to the virus detection.
Table 1 response droping frequency and SARS antibody concentration relation table
| SARS antibody concentration (mg/mL) | 37.50 | 17.75 | 15.00 | 12.50 |
| To PBS droping frequency Δ f 1(ΔHz) | Exceed survey frequency | 4018 | 1320 | 1880 |
| To SARS virus droping frequency Δ f 2(ΔHz) | 5209 | 4050 | 2809 | |
| f 2-f 1(ΔHz) | 1191 | 2730 | 929 |
As can be seen from the above table,, can't detect, when antibody concentration is 17.75mg/mL, the response of virus be reached 5709Hz when phenomenon frequently takes place during for 37.5mg/mL disorderly antibody concentration, but to background PBS signal also up to 4018Hz, background signal is bigger.After taking all factors into consideration the deduction background, the SARS antibody concentration is preferable with 15.00mg/mL, and its response signal to virus can reach 2700Hz.
The working curve that the SARS piezoeletric quartz sensor detects SARS virus among the PBS:
Preparation contains the PBS aqueous solution of SARS virus of variable concentrations, and each sample size is 1 μ L, detects virus concentration respectively and be 5,3,2,1.5 and the response signal during 1mg/mL, deduction PBS background signal, and the response curve of virus is as shown in figure 12.
For testing SA RS piezoeletric quartz sensor reaches the precision that virus is detected to water, under same testing conditions, use same sensor that the virus (1mg/mL) and the PBS aqueous solution of same concentrations are carried out ten experiments, the result is shown in table 2 and table 3.
The same wafer of table 2 is to the detection precision of PBS aqueous solution
| Number of | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | X | δ | RSD |
| Response signal (Hz) | 2277 | 2071 | 2518 | 1859 | 1901 | 2338 | 2579 | 2439 | 1933 | 2038 | 2195 | 268 | 0.1221 |
The same wafer of table 3 is to the detection precision of same concentration virus among the PBS
| Number of | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | X | δ | RSD |
| Response signal (Hz) | 2823 | 2695 | 3164 | 2475 | 2503 | 3194 | 3035 | 2959 | 2430 | 2549 | 2783 | 278 | 0.0998 |
As can be seen from the above table, the relative standard deviation that PBS is detected for ten times is 12.21%, and the relative standard deviation that virus is detected ten times is 9.98%.Calculate according to S/N=2, minimum detectability is 0.61 μ L.
For the collimation that the test piezoeletric quartz sensor detects, in identical conditions, use different piezoeletric quartz sensors that the virus (1mg/mL) and the PBS aqueous solution of same amount are carried out ten experiments, the result is as shown in table 4.
Table 4 different chips is to the detection collimation (Hz) of PBS and SARS poison wherein
| Sequence number | Fundamental frequency | Δ f behind the glair A | Be coated with Δ f behind the antibody | Response to PBS | Contain the PBS response of virus | The |
| 1 | 9013354 | 12785 | 25160 | 1853 | 2393 | 540 |
| 2 | 8998427 | 12191 | 23806 | 1671 | 2374 | 703 |
| 3 | 9010992 | 12937 | 27136 | 2206 | 2708 | 502 |
| 4 | 9002158 | 11838 | 20513 | 2174 | 2718 | 544 |
| 5 | 9015014 | 11588 | 25308 | 1848 | 2549 | 701 |
| 6 | 9013634 | 12835 | 28679 | 1950 | 2400 | 450 |
| 7 | 8990958 | 12167 | 28251 | 2258 | 2889 | 631 |
| 8 | 9008701 | 12166 | 27581 | 2264 | 2916 | 652 |
| 9 | 9002316 | 12280 | 24278 | 1954 | 2407 | 453 |
| 10 | 9016836 | 13288 | 27652 | 2240 | 2885 | 645 |
| X | 9007239 | 12408 | 25836 | 2042 | 2624 | 582 |
| RSD | 0.0430 | 0.0979 | 0.1041 | 0.0860 | 0.1657 |
As can be seen from the above table, when preparing ten different sensors, frequency change relative standard deviation behind the glair A is 4.30%, the frequency change relative standard deviation that is coated with behind the antibody is 9.79%, sensor is 10.41% to the relative standard deviation that PBS ten times detects, and is 8.60% partially to the relative standard of the PBS aqueous assay that contains same concentrations virus ten times.Relative standard deviation to the virus response behind the deduction background is 16.57%.Relative standard deviation is all less than 20%.
The SARS piezoeletric quartz sensor is to the virus response of homophyletic not:
Up to now, found the SARS virus strain that kind is different surplus in the of ten in the world wide, domestic also have multiple, as No. 01,02,03, Beijing, No. 01,02,03, Guangzhou etc.SARS piezoeletric quartz sensor of the present invention is to induce the horse serum antibody that gets to develop according to No. 01 Strain in Beijing, for testing of the response of this sensor to different strain virus, Figure 13 be piezoeletric quartz sensor to No. 01 viral PBS aqueous solution in Guangzhou (1.88mg/mL) test result, frequency change is 2903Hz.As shown in figure 14, same sensor is 2209Hz to same amount PBS test frequency changing value under the same conditions.From two figure relatively, frequency-splitting is 694Hz.And on No. 01 SARS virus adsorption curve in Beijing as can be known, the frequency-splitting that 1.88mg/mL virus can cause is 1602Hz.This illustrates that this sensor can be used to detect No. 01 SARS virus in Guangzhou, but response is lower than the detection to No. 01 virus in Beijing.
Whether can cause interference to the measurement of SARS piezoeletric quartz sensor in order to study other viruses, the present inventor tests with the A1 influenza virus, and to the experimental result of influenza virus as shown in figure 15, response signal is 1853Hz.Figure 16 is the detection curve of SARS piezoeletric quartz sensor to PBS, and response signal is 1850Hz.Hence one can see that, and influenza virus is to detecting not obviously influence.
The SARS piezoelectric immunosensor is to the testing result of SARS virus in saliva and the saliva:
For more same SARS piezoeletric quartz sensor to saliva and contain the testing result of saliva of virus, done following experiment.Figure 17 is the response curve of SARS immunosensor to saliva, its frequency decline 2520Hz, (solvent is same people's a saliva to Figure 18 to virus for same sensor under the same terms, concentration is 4mg/mL) response curve, its frequency decline 5758Hz, deduction saliva background, the frequency that causes by virus and the antibodies 3238Hz for this reason that descends, illustrate that response signal is obvious, the response time is about 1 minute.
Figure 19 is the working curve of same sensor to the SARS virus (same people's saliva) of variable concentrations, and each sample size is 1 μ L, and the concentration of virus is respectively 4,3,2,1,0.5mg/mL, deduction saliva background signal.
For the precision that testing sensor detects, in identical conditions, use same sensor that the virus (1mg/mL) and the saliva of the same concentration of same people's saliva preparation are carried out ten detections, the result is shown in table 5, table 6.
The same wafer of table 5 is to the detection precision of saliva
| Number of | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | X | δ | RSD |
| Response signal (Hz) | 1825 | 2231 | 2469 | 2160 | 2453 | 2057 | 2433 | 2109 | 1827 | 1870 | 2143 | 254 | 0.1096 |
The same wafer of table 6 is to the detection precision of 1mg/mL virus in the saliva
| Number of | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | X | δ | RSD |
| Response signal (Hz) | 2539 | 2821 | 3051 | 2900 | 3205 | 2539 | 3312 | 2879 | 2522 | 2422 | 2819 | 309 | 0.1183 |
As can be seen from the above table, the relative standard deviation that PBS is detected for ten times is 1.22%.The relative standard that virus is detected for ten times is 10.39% partially.Calculate according to S/N=2, minimum detectability is 0.6 μ g/ μ L.
For the collimation that testing sensor detects, in identical conditions, use different sensors that the virus (1mg/mL) and the saliva background of same concentrations in the saliva are carried out ten experiments, the result is as shown in table 7.
Table 7 different chips is to the detection collimation (Hz) of virus in saliva and the saliva
| Sequence number | Fundamental frequency | Δ f behind the glair A | Be coated with Δ f behind the antibody | The saliva response | Virus response in the saliva | The |
| 1 | 9013354 | 12785 | 25160 | 2219 | 2874 | 655 |
| 2 | 8998427 | 12191 | 23806 | 1748 | 2510 | 762 |
| 3 | 9010992 | 12937 | 27136 | 1757 | 2641 | 884 |
| 4 | 9002158 | 11838 | 20513 | 2420 | 2959 | 539 |
| 5 | 9015014 | 11588 | 25308 | 2538 | 3173 | 635 |
| 6 | 9013634 | 12835 | 28679 | 1844 | 2334 | 490 |
| 7 | 8990958 | 12167 | 28251 | 1813 | 2547 | 734 |
| 8 | 9008701 | 12166 | 27581 | 1974 | 2574 | 600 |
| 9 | 9002316 | 12280 | 24278 | 1751 | 2448 | 697 |
| 10 | 9016836 | 13288 | 27652 | 2006 | 2667 | 661 |
| X | 9007239 | 12408 | 25836 | 2007 | 2673 | 666 |
| RSD | 0.0430 | 0.0979 | 0.1446 | 0.0959 | 0.1697 |
As can be seen from the above table, the relative standard deviation that saliva is detected for ten times is 14.46%.The relative standard deviation that the saliva that contains virus is detected ten times is 9.59%.Relative standard deviation to the virus response is 16.97%.
Same SARS piezoelectric immunosensor detects 50 people's saliva.Population mean is 2128Hz; The male sex 37 people wherein, mean value is 2070Hz; Women 13 people, mean value is 2298Hz.From age level, its testing result is as shown in table 8.
Table 8 all ages and classes stage experimenter testing result
| Age | 0-10 | 10-20 | 20-30 | 30-40 | 40-50 | 50-60 | 60-70 |
| | 6 | 3 | 21 | 6 | 3 | 3 | 8 |
| Mean value (Hz) | 2464 | 1934 | 2030 | 2028 | 2364 | 2379 | 2102 |
Figure 20 is the different crowd's saliva response distribution plan of age, and from Figure 20 as seen, response is between 1600-2700Hz, and most responses are between 1700-2500Hz (84%).
Cold compartment of refrigerator (4-6 ℃) is put in the sealing of SARS piezoeletric quartz sensor preserved, tested its response to virus (concentration is 4mg/mL) and PBS background every 5 days, test result sees Table 9.
Table 9 sensor active testing result
| Standing time (my god) | 0 | 5 | 10 | 15 | 20 | 25 | 30 |
| To viral response frequency (Δ Hz) | 5912 | 5984 | 5400 | 5672 | 5419 | 5295 | 4976 |
| To PBS response frequency (Δ Hz) | 2300 | 2343 | 2060 | 2376 | 2277 | 2195 | 1998 |
| Response change | 3612 | 3641 | 3440 | 3296 | 3142 | 3100 | 2978 |
As seen from the above table, the antibody stable in properties with a-protein after fixing, activity is not seen obvious decline after one month.
The SARS piezoeletric quartz sensor can repeatedly use, and generally can use continuously tens of times.But to fully resolve after each the use, generally be advisable to be resolved to its fundamental frequency after filming.
Can draw to draw a conclusion from above-mentioned experimental result:
1, uses above-mentioned ultrasonic atomizatio method, SARS virus in 1 μ L PBS aqueous solution and the saliva can be atomized fully.
2, with the albumin A be crosslinking chemical, the piezoelectric immunosensor made from horse serum SARS antibody can fast detecting water outlet in about 2 minutes, contained SARS virus in the saliva.SARS virus detection among the PBS is limited to 0.6 μ g/ μ L, SARS virus in the saliva is detected be limited to 0.6 μ g/ μ L.
3, piezoeletric quartz sensor has extremely strong specificity, and requires antibody that higher tiring arranged.Description of test obtains 12 SARS rehabilitation patient serum from SARS fixed hospital, Xiao Tang mountain, therefrom uses (NH
4)
2SO
4The precipitation method are extracted and are obtained antibody, and the piezoeletric quartz sensor made from this antibody does not have response to SARS virus.And the horse serum antibody of directly inducing generation with this SARS virus is because of having very high tiring, and the piezoeletric quartz sensor of making is good to SARS virus testing result description effect.
4, with of the 50 people saliva testing results explanation of same piezoeletric quartz sensor to all ages and classes section, all ages and classes, the people's of different physiological situations saliva testing result is discrepant.But 84% manual inspection measured value is between 1700Hz-2500Hz (detection mean value is 2128Hz).
5, same piezoeletric quartz sensor is to same people's the parallel detection of saliva ten times, and it is 11.83% that presentation of results detects relative standard deviation.Ten piezoeletric quartz sensors illustrate same people's saliva testing result, though sensor fundamental frequency and thickness difference slightly, its testing result deviation is within 20% scope.
6, because of water has interference to detection, the storage of SARS piezoeletric quartz sensor should exist lower seal to preserve in the reefer of refrigerator at silica gel or other drying agent etc.Before the use, should in measurement environment, place a period of time, make that the micromolecule in the antibody film and surrounding air reaches complete equipilibrium on the sensor, re-use after the frequency stabilization.
7, piezoeletric quartz sensor is reusable tens of times, but should fully resolve before each the use, makes it return to fundamental frequency, and the parsing time was advisable with ten minutes.
9, the piezoeletric quartz sensor made of the horse serum polyclonal antibody of inducing with No. 01 SARS virus in Beijing not only can detect No. 01 SARS virus in Beijing, and can detect No. 01 SARS virus in Guangzhou of homophyletic not.
Claims (9)
1, a kind of viral detection system comprises detection system, it is characterized in that: also comprise atomization system and warning system; Described atomization system links to each other with described warning system by described detection system;
Described atomization system comprises ultrasonic atomizatio generating means, ultrasonic transduction device and atomizing control circuit; Described detection system comprises mass measurement formula immunosensor and testing circuit device; Described warning system is the single-chip microcomputer warning system; Described single-chip microcomputer warning system comprises difference frequency device circuit, single-chip microcomputer treatment circuit and warning circuit.
2, viral detection system according to claim 1 is characterized in that: described ultrasonic atomizatio generating means is ultrasonic logic shaker; Described ultrasonic transduction device is the piezoelectric ceramic ultrasonic oscillation chip; Described ultrasonic logic shaker links to each other with described piezoelectric ceramic ultrasonic oscillation chip by described atomizing control circuit; Described mass measurement formula immunosensor is the piezoeletric quartz sensor that scribbles the antibody film, and two electrode inserts on the described testing circuit device; Place described piezoelectric ceramic ultrasonic oscillation chip on the support of the described piezoeletric quartz sensor top that scribbles the antibody film; Described piezoeletric quartz sensor that scribbles the antibody film and described piezoelectric ceramic ultrasonic oscillation chip and be placed in the closed container; The described piezoeletric quartz sensor that scribbles the antibody film links to each other with warning circuit with described difference frequency device circuit, single-chip microcomputer treatment circuit.
3, a kind of method for detecting virus utilizes the described viral detection system of claim 1 to carry out, and its step is as follows:
(1) gets the sample that contains virus,, extract, be mixed with solution through handling;
(2) get above-mentioned solution, drop to the ultrasonic atomizatio generating means;
(3) the ultrasonic transduction device transmits energy to described ultrasonic atomizatio generating means, and the solution on it is carried out ultrasonic atomizatio;
(4) mass measurement formula immunosensor and the testing circuit device that scribbles the antibody film is installed in the closed container, the virus of aerosolization is deposited on the chip of described mass measurement formula immunosensor;
(5) described mass measurement formula immunosensor links to each other with described warning circuit, and the virus quantity that is adsorbed acquires a certain degree, and warning device promptly sends alerting signal.
4, method for detecting virus according to claim 3 is characterized in that: described mass measurement formula immunosensor is the piezoeletric quartz sensor that scribbles the antibody film.
5, method for detecting virus according to claim 4 is characterized in that: described ultrasonic atomizatio generating means is ultrasonic logic shaker; Described ultrasonic transduction device is the piezoelectric ceramic ultrasonic oscillation chip.
6, method for detecting virus according to claim 5 is characterized in that: described virus is SARS virus.
7, method for detecting virus according to claim 6 is characterized in that: described ultrasonic transduction device is the electric current of 440mA to the transmission of described ultrasonic atomizatio generating means; The duration of oscillation of described piezoelectric ceramic ultrasonic oscillation chip is 0.09 second, and vibration is spaced apart 0.2 second, and the number of oscillation is 15 times.
8, according to each described method for detecting virus among the claim 3-7, it is characterized in that: the described Virus Sample consumption that contains is 0.5-1.5 μ L.
9, the method for detecting virus described in according to Claim 8, it is characterized in that: the described concentration that contains Virus Sample is 0.6-4 μ g/ μ L.
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| EP2634580A4 (en) * | 2010-10-29 | 2014-04-30 | Tokyo Electron Ltd | Virus detection device and virus detection method |
| US8911955B2 (en) | 2010-10-29 | 2014-12-16 | Tokyo Electron Limited | Virus detection device and virus detection method |
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