The aerocolloidal system and method for a kind of real-time monitoring bio
Technical field
The present invention relates to a kind of bioaerosol real-time monitoring system and method, mainly can be used for monitoring in real time airborne pathogenic former (comprising virus, bacterium, anaphylactogen etc.), thereby when taking precautions against bio-hazard such as large-scale influenza epidemic situation, bio-terrorism activity, play a role, also can be used for dynamically characterizing the variation of air microorganism etc.
Background technology
The breathing of bioaerosol exposes and has caused a lot of health problems, and large-scale in recent years flu outbreak has caused huge lives and properties and financial loss such as the influenza of the H1N1 of SARS in 2003 and 2009.In order to take precautions against and resist these danger better, press for a kind of can be to virus, the bacterium technology of monitoring in real time in the air, yet this is a technical barrier for a long time.In the past few years a lot of technology were once attempted being used for realizing the on-line monitoring of bioaerosol, comprising mass-spectrometric technique, surface enhanced Raman technology, cell streaming instrument, also have based on the Ultraviolet Aerodynamic ParticleSizer (UVAPS) of fluorescence etc., but these technology are difficult to realize to the examination of microorganism in the air or exist very high problems such as false positive.Though the appearance of gene amplification makes the accuracy of the airborne microorganism of monitoring improve greatly, these systems are difficult to and Sampling techniques etc. are integrated into automatic monitoring system.
Performances such as the power of the uniqueness that nano material/structural table reveals, electricity, heat, light, magnetic, can realize the performance boundary of new function or breakthrough conventional device, realize having the nano-device and the system of new principle or high-performance (as highly sensitive, reduce power consumption, lower noise).Silicon nanowires is a kind of as one dimension Nano structure, has as many excellent properties different with the body silicon materials such as electron transport, field emission characteristic, surfactivity and quantum limit, thereby has very application prospects aspect the making of low-dimensional nano-device.Can control its semi-conductor conductivity effectively by mixing.In recent years, the field-effect transistor based on nano wire successfully has been applied in the influenza virus and several bioproteins of parallel monitoring of monitoring in the water.But these researchs are primarily aimed at the microorganism monitoring in the liquid medium, also do not report the research that utilizes silicon nanowires field-effect transistor on-line monitoring bioaerosol at present.
Summary of the invention
At the problem of above-mentioned existence, the present invention is intended to by technology such as integrated bio sampling of aerosol, microflow control technique and biological detection, feeble signal amplifications, realizes the former on-line monitoring that causes a disease in the air has been filled up the blank in this field.
Technical scheme provided by the invention is as follows:
Scheme 1: the aerocolloidal system of a kind of real-time monitoring bio (as shown in Figure 1), it is characterized in that, comprise three parts:
-sample collecting and supply unit comprise sampling thief 1, sample delivery pipe 2, peristaltic pump 9, collection containers 10 and sampling media container 11; The conveying of sample is finished in the collection that described sampling thief 1 is realized bioaerosol then by sample delivery pipe 2 under the effect of peristaltic pump 9 by sampling media, and through micro-fluidic 4, biochip 3, be transported to collection containers 10 at last;
-signal detection and processing unit comprise prime amplifier 6, lock-in amplifier 7, are integrated with the biochip 3 and micro-fluidic 4 of silicon nano-wire biological sensor 5;
Output of-data and display unit comprise the host computer and the indicating meter that link to each other with lock-in amplifier 7; Be equipped with on the described host computer and be used for the data that lock is put are transmitted the software that control, signal post-processing and result show.
Scheme 2: a kind of preferred realization as scheme 1 is characterized in that described sampling thief 1 is electrostatic field sampling thief (as shown in Figure 3), comprising: dome electrode 101, circular electrode 102, sample collecting groove 104; Wherein, dome electrode 101 is fixed on the insulator foot 106, and circular electrode 102 is positioned at the sphere center position of insulator foot 106 first ball shaped electrodes 101, and described sample collecting groove 104 is positioned at the top of circular electrode 102; A plurality of air intlets 111 are arranged at dome electrode 101 tops; On insulator foot 106, be provided with two airs 110; In sample collecting groove 104, there is 108 and one of a sample delivery outlet to gather liquid input aperture 109.
Scheme 3: as a kind of preferred realization of scheme 1, it is characterized in that, in described signal detection and the processing unit, the microelectrode of metal probe contact devices, connect input electrical signal by the metal probe lead-in wire and be applied on the chip electrode, draw detected signal simultaneously and input to surveying instrument (prime amplifier 6 and lock-in amplifier 7); Wherein, two metal probes connect the source-drain electrode of some silicon nano-wire biological sensors respectively, be connected with cable between metal probe and lock-in amplifier 7 and the prime amplifier 6, a metal probe links to each other with the reference output of lock-in amplifier 7, and another probe output is connected to the voltage input of lock-in amplifier 7 through prime amplifier 6.With reference to the output signal maximum amplitude is 5V, and highest frequency is 100kHz.
Scheme 4: a kind of preferred realization as scheme 1, it is characterized in that the electrode surface material of described biochip 3 is Au, for reducing the contact resistance between probe and microelectrode, selecting the probe tip material for use is the composition metal of Au.
Scheme 5: a kind of preferred realization as scheme 4 is characterized in that the needle point radius-of-curvature of described probe tip is 25 μ m.
Scheme 6: a kind of preferred realization as scheme 2 is characterized in that, in described sample collecting and the supply unit, the pump head of peristaltic pump 9 is two-tube output, one end of one of them flexible pipe connects sampling media (for example, sterilized water), and the other end connects sample collecting groove 104 input interfaces of sampling thief 1; One end of another flexible pipe connects sample collecting groove 104 output interfaces, and the other end is connected on the inlet of micro-fluidic 4 on the biochip 3.Certain rotating speed is set, in the peristaltic pump operational process air sample that collects is transported to biochip surface from the collection groove of sampling thief by micro-fluidic 4, by micro-fluidic 4 outlet sample delivery is arrived sample collecting bottle 10 at last, simultaneously the sampling media of gathering in the groove is replenished.Can change the speed that sample flow is crossed chip by the rotating speed of regulating peristaltic pump.
Scheme 7: as a kind of preferred realization of scheme 1, it is characterized in that, in described data output and the display unit, in order to show detection signal in real time, consider follow-up possible multi-channel detection simultaneously, adopt extendible GPIB card and private cable to connect lock-in amplifier 7 and host computer; Cable links to each other with the RS232 serial ports of lock-in amplifier 7; On the described host computer computer software is installed, this software realizes that Data Transmission Controlling, signal post-processing, result that lock is put show; Program interface comprises the input reference voltage value, sampling number/sampling time, and magnification, gauge dial shows and the measured electricity in back that converts is led change curve, and can preserve the data that real-time monitoring is surveyed.
Scheme 8: a kind of preferred realization as scheme 7 is characterized in that described computer software has the function that triggers warning when detecting thing to be checked.Even under unattended situation, also can realize real-time monitoring like this to microorganism.
The present invention provides a kind of method of utilizing scheme 1 described system to monitor in real time simultaneously, and scheme is as follows:
Scheme 9: a kind of scheme 1 described system that utilizes carries out the aerocolloidal method of real-time monitoring bio, it is characterized in that, comprises the steps:
A), biochip 3 is carried out specific antibody modify at the object microorganism A that is monitored (for example: virus, bacterium, anaphylactogen etc.);
B) start-up system realizes the continuous sampling of environmental sample, the real-time conveying of sample, the real-time demonstration of silicon nanowires conductivity;
C) when detected object microorganism A is arranged in the air sample that collects, in indicating system, demonstrate the electroconductibility generation obvious variation of silicon nanowires, the electroconductibility that is higher than the correspondence of silicon nanowires itself or negative air sample far away, based on the specificity of antibody, the phenomenon that electric conductivity value significantly changes can confirm the existence of detected object microorganism A.
Scheme 10: a kind of preferred realization as scheme 9, it is characterized in that, after detecting microorganism A existence, report to the police by launch computer.Warning in the time of can realizing unmanned like this.
Beneficial effect of the present invention: this invention provides the novel method in bioaerosol on-line monitoring field, realized utilizing biochip on-line monitoring bioaerosol first, monitoring time is significantly shorter than existing most of technology, realized real-time monitoring to influenza virus in the air, not only can screen kind, but also can be quantitative.In medical and health organization, drome, critical point, airport and strick precaution influenza and bio-terrorism activity, there is important use to be worth.
Description of drawings
The online bioaerosol Monitoring systems of Fig. 1 structural representation.
The airborne influenza virus of Fig. 2 on-line monitoring (H3N2 hypotype) sample.
Fig. 3 electrostatic field sampling thief structural representation.
Wherein, mark is described as follows among Fig. 1:
1: the electrostatic field sampling thief |
2: the sample delivery pipe |
3: biochip |
4: micro-fluidic |
5: biosensor (integrated a plurality of silicon nanowires on it) |
6, prime amplifier |
7, lock-in amplifier |
8, display routine |
9, peristaltic pump |
10, sample collection bottle |
11, sampling media container |
12, vacuum pump |
13, high-voltage DC power supply |
|
Mark is described as follows among Fig. 3:
101: dome electrode |
102: circular electrode |
103: the impressed voltage interface |
104: the sample collecting groove |
105: particle charger |
106: insulator foot |
107: the retaining screw mother |
108: the sample delivery outlet |
109: gather the liquid input aperture |
110: air |
111: air intlet |
|
Embodiment
With reference to structure shown in Figure 1, the present invention has realized a concrete system: electrostatic field sampling thief 1 connects biochip 3 by sample delivery pipe 2, microfluidic channel 4 is arranged on biochip 3, integrated a plurality of silicon nano-wire biological sensors 5 on it, on biochip 3, also be connected with signal preamplifier 6 and signal lock-in amplifier 7 (signal preamplifier 6 also links to each other with signal lock-in amplifier 7), computer LabView signal display routine 8 is installed on the signal lock-in amplifier 7, is used to show monitoring result; Electrostatic field sampling thief 1 links to each other with a peristaltic pump 9 by another root sample delivery pipe 2, in order to realize the real-time transmission of sampling media; The two ends of peristaltic pump 9 connect sample collection bottle 10 and sample solution bottle 11 respectively; On the base of electrostatic field sampling thief, be connected to a vacuum pump 12, in order to drive the circulation of air in sampling thief; Electrostatic field sampling thief 1 links to each other with high-voltage DC power supply 13, in order to drive sampling thief work.
Described electrostatic field sampling thief 1 is a kind of air sampler based on electrostatic field (as shown in Figure 3), it is characterized in that, comprising: dome electrode 101, circular electrode 102, sample collecting groove 104; Wherein, dome electrode 101 is fixed on the insulator foot 106, and circular electrode 102 is positioned at the sphere center position of insulator foot 106 first ball shaped electrodes 101, and described sample collecting groove 104 is positioned at the top of ball shaped electrode 102; A plurality of air intlets 111 are arranged at dome electrode 101 tops; On insulator foot 106, be provided with a plurality of airs 110; In sample collecting groove 104, there is 108 and one of a sample delivery outlet to gather liquid input aperture 109.103 is the impressed voltage interface among the figure, and 105 is particle charger, and 107 for being fixed to dome electrode 101 on the nut on the insulator foot 106.This electrostatic field sampling thief utilizes electrostatic field to make in the sample space in the air electrically charged microorganism under electric field action, concentrates along direction of an electric field to be deposited on the hemisphere center relatively in the liquid medium than the zonule, thereby realizes the purpose of sampling.
Described signal preamplifier 6 is LI76 (NF), and signal lock-in amplifier 7 is LI5640 (NF), and vacuum pump 12 is the SKC pump, and biochip 3 is made of silicon nanowire array, and data-interface is AgilentPCI-GPIB82350B.
The electrode surface material of sensor chip 5 is Au, and for reducing the contact resistance between probe and microelectrode, selecting the needle point material for use is the compound Au probe (the needle point radius-of-curvature is 25 μ m) of Au.Be connected with the bnc interface cable between metal probe and lock-in amplifier and the prime amplifier.One of them metal probe links to each other with the reference output of lock-in amplifier, and another metal probe is connected to the voltage input of lock-in amplifier through prime amplifier.With reference to the output signal maximum amplitude is 5V, and highest frequency is 100kHz.
Monitoring method following (is example with influenza virus H3N2):
(1), biochip is carried out specific antibody (H3N2 Antibody of Influenza) modify at first at the object of being monitored (H3N2);
(2) start-up system realizes the continuous sampling of environmental sample, the real-time conveying of sample, the real-time demonstration of silicon nanowires conductivity;
(3) when in the air sample that collects influenza virus being arranged, obvious variation can take place in the electroconductibility of silicon nanowires, the electroconductibility that is higher than the correspondence of silicon nanowires itself or negative air sample far away, based on the specificity of antibody, the phenomenon that electric conductivity value significantly changes can confirm the existence of influenza virus.
Biochip is carried out specific antibody (H3N2 Antibody of Influenza) modify, mainly finish by 4 steps: the first step, under the room temperature, nano-device and glutaraldehyde (5% glutaraldehyde, pH=8) reaction is 1 hour, and (10mM, pH=8) flushing is 5 minutes to use phosphate buffered saline buffer again.In second step, H3N2 antibody (0.1mg/ml antibody-solutions, pH=8 comprise 4mM sodiumcyanoborohydride) wraps by nano-device, and reaction conditions is 4 ℃, 14 hours.In this process, the aldehyde groups combination on the amino group of antibody end and nano-device surface.In the 3rd step, (10mM, pH=8) flushing is 5 minutes with phosphate buffered saline buffer.In the 4th step, (the 100mM Tri N-Propyl Amine pH=8) wraps by nano-device 2 hours time to utilize Tri N-Propyl Amine solution.Utilize the amino group sealing nano-device surface of Tri N-Propyl Amine not have aldehyde groups with antibodies.After corresponding microorganism occurring in the air, it is condensing to be sampled the device collection, enters the chip detection part, and Ag-Ab immunity association reaction takes place, and the bio signal of appearance is converted to electrical signal through preposition amplification and phase-locked amplification, has promptly realized on-line monitoring.
Fig. 2 is the partial data of on-line monitoring influenza virus (H3N2 hypotype), according to the information among the figure, when having influenza virus (H3N2 hypotype) in the air, total system can show that in 1-3 branch clock time the significant electricity of silicon nanowires leads variation, meanwhile, result according to gene amplification shows that the sample correspondence that influenza virus concentration is high in the air higher silicon nanowires electroconductibility.This system not only can be used for monitoring virus, and can carry out on-line monitoring to biotic components such as airborne bacterium and anaphylactogens equally.On detection time,, usually need 2-3 hour such as gene amplification considerably beyond other conventional art.
To sum up, the present invention has following advantage:
(1) the present invention's integrated by to technology such as silicon nano-wire biological sensor, micro-fluidic, air continuous sampling and the phase-locked amplifications of signal, formed the bioaerosol real-time monitoring system, result of study shows that when having influenza virus to exist in the air, this system can show early warning signal in 1-3 minute;
(2) this system integration the technology of different field comprise the amplification and the demonstration of environment sampling, micro-fluidic delivery system, nanotechnology and detection signal;
(3) this system is an auto-real-time monitoring system that does not need artificial supervision, can realize the real-time examination of living species in the air and quantitatively.