CN102174382B - System and method for monitoring bioaerosol in real time - Google Patents
System and method for monitoring bioaerosol in real time Download PDFInfo
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Abstract
The invention discloses a system and method for monitoring bioaerosol in real time. The system comprises a sample acquisition and transmission unit, a signal detection and processing unit and a data output and display unit, wherein, the sample acquisition and delivery unit comprises a sampler (1), a sample delivery pipe (2), a peristaltic pump (5) and a sampling medium container; the signal detection and processing unit comprises a preamplifier (6), a phase-locked amplifier (7) and a biological chip (3) integrated with a biosensor (5); and the data output and display unit comprises a computer host and a display connected with the phase-locked amplifier (7). The system and method provided by the invention can be used for monitoring infectious agents in the air, comprising viruses, bacteria, and allergen and the like in real time, can play a role in preventing the biological risks, such as large flu epidemic situation and bioterrorism activity and the like, and also can be used for dynamically representing the changes of microorganism in the air and the like.
Description
Technical field
The present invention relates to a kind of bioaerosol real-time monitoring system and method, mainly can be used for Real-Time Monitoring airborne pathogenic former (comprising virus, bacterium, anaphylactogen etc.), thereby play a role when the bio-hazard such as the large-scale Influenza epidemic situation of strick precaution, bio-terrorism activity, also can be used for the variation of dynamic characterization microbes in air etc.
Background technology
The breath exposure of bioaerosol has caused a lot of health problems, and large-scale flu outbreak has in recent years caused huge lives and properties and financial loss such as the influenza of the H1N1 of the SARS of 2003 and 2009.In order to take precautions against better and resist these danger, can carry out to virus, bacterium in air the technology of Real-Time Monitoring in the urgent need to a kind of, yet this is individual 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, the Raman technology that the surface strengthens, Flow cytometry, also have based on the Ultraviolet Aerodynamic ParticleSizer (UVAPS) of fluorescence etc., but these technology are difficult to realize to the examination of microbes in air or exist the very high problems such as false positive.Although the appearance of gene amplification makes the accuracy of the airborne microorganism of monitoring greatly improve, these systems are difficult to and Sampling techniques etc. are integrated into automatic monitoring system.
The 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 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 from the body silicon materials such as electron transport, field emission characteristic, surfactivity and quantum limit, thereby has application prospect very widely aspect the making of low-dimensional nano-device.Can effectively control its semi-conductor conductivity by doping.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 water.But these researchs also do not report mainly for the microorganism monitoring in liquid medium the research that utilizes silicon nanowires field-effect transistor on-line monitoring bioaerosol at present.
Summary of the invention
For 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 air has been filled up the blank in this field.
Technical scheme provided by the invention is as follows:
Scheme 1: a kind of system of Real-Time Monitoring bioaerosol (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; Then the collection that described sampling thief 1 is realized bioaerosol completes the conveying of sample by sample delivery pipe 2 under the effect of peristaltic pump 9 by sampling media, and through micro-fluidic 4, biochip 3, be transported at last collection containers 10;
-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;
The output of-data and display unit comprise the host computer and the indicating meter that are connected with lock-in amplifier 7; Be equipped with on described host computer for the data that lock is put and transmit the software that control, signal post-processing and result show.
Scheme 2: a kind of preferred realization as scheme 1, it 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 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; Be provided with two airouts 110 on insulator foot 106; There are 108 and one of sample delivery outlets to gather liquid input aperture 109 in sample collecting groove 104.
Scheme 3: as a kind of preferred realization of scheme 1, it is characterized in that, in described signal detection and processing unit, the microelectrode of metal probe contact devices, connect input electrical signal by the metal probe lead-in wire and be applied on chip electrode, draw simultaneously detected signal and input to surveying instrument (prime amplifier 6 and lock-in amplifier 7); Wherein, two metal probes connect respectively the source-drain electrode of some silicon nano-wire biological sensors, be connected with cable between metal probe and lock-in amplifier 7 and prime amplifier 6, a metal probe is connected 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.Be 5V with reference to the output signal maximum amplitude, 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 is the composition metal of Au.
Scheme 5: a kind of preferred realization as scheme 4, it is characterized in that, the needle point radius-of-curvature of described probe tip is 25 μ m.
Scheme 6: as a kind of preferred realization of scheme 2, it is characterized in that, in described sample collecting and supply unit, the pump head of peristaltic pump 9 is two-tube output, one end of one of them flexible pipe (for example connects sampling media, sterilized water), 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 entrance of micro-fluidic 4 on biochip 3.Certain rotating speed is set, in the peristaltic pump operational process, the 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 that gathers in groove is replenished.Can change by the rotating speed of regulating peristaltic pump the speed that sample flow is crossed chip.
Scheme 7: as a kind of preferred realization of scheme 1, it is characterized in that, in described data output and display unit, in order to show in real time detection signal, consider simultaneously follow-up possible multi-channel detection, adopt extendible GPIB card and private cable to connect lock-in amplifier 7 and host computer; Cable is connected with the RS232 serial ports of lock-in amplifier 7; On 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 rear measured electricity 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, it is characterized in that, described computer software has the function of trigger alarm when thing to be checked being detected.Even also can realize like this Real Time Monitoring to microorganism in unattended situation.
The present invention provides a kind of method of utilizing scheme 1 described system to carry out Real Time Monitoring simultaneously, and scheme is as follows:
Scheme 9: a kind of method of utilizing scheme 1 described system to carry out the Real-Time Monitoring bioaerosol, it is characterized in that, comprise the steps:
A) for the object microorganism A that monitors (such as virus, bacterium, anaphylactogen etc.), biochip 3 is carried out specific antibody modify;
B) startup 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, the electroconductibility that demonstrates silicon nanowires in indicating system occurs significantly to change, far away higher than the electroconductibility of the correspondence of silicon nanowires itself or negative air sample, 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 microorganism A existence being detected, reported 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 first utilizing biochip on-line monitoring bioaerosol, monitoring time is significantly shorter than existing most of technology, realized the Real-Time Monitoring to influenza virus in 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, important using value is arranged.
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 sampler structure schematic diagram.
Wherein, in Fig. 1, mark is described as follows:
| 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, |
| 7, lock-in |
8, |
| 9, |
10, |
| 11, |
12, |
| 13, high-voltage DC power supply |
In Fig. 3, mark is described as follows:
| 101: dome electrode | 102: circular electrode |
| 103: the impressed voltage interface | 104: the sample collecting groove |
| 105: particle charger | 106: insulator foot |
| 107: retaining screw is female | 108: the sample delivery outlet |
| 109: gather the liquid input aperture | 110: airout |
| 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, also be connected with signal preamplifier 6 and signal locking amplifier 7 (signal preamplifier 6 also is connected with signal locking amplifier 7) on biochip 3, computer LabView signal display routine 8 is installed on signal locking amplifier 7, is used for showing monitoring result; Electrostatic field sampling thief 1 is connected 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 respectively sample collection bottle 10 and sample solution bottle 11; Be connected to a vacuum pump 12 on the base of electrostatic field sampling thief, in order to drive the circulation of air in sampling thief; Electrostatic field sampling thief 1 is connected 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 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; Be provided with a plurality of airouts 110 on insulator foot 106; There are 108 and one of sample delivery outlets to gather liquid input aperture 109 in sample collecting groove 104.In figure, 103 is the impressed voltage interface, and 105 is particle charger, and 107 for being fixed to dome electrode 101 on the nut on insulator foot 106.This electrostatic field sampling thief utilizes electrostatic field to make in the sample space in air electrically charged microorganism under electric field action, concentrates in the liquid medium that is deposited on half ball center's less zone along direction of an electric field, thereby realizes the purpose of sampling.
Described signal preamplifier 6 is LI76 (NF), and signal locking 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 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 prime amplifier.One of them metal probe is connected 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.Be 5V with reference to the output signal maximum amplitude, highest frequency is 100kHz.
Monitoring method following (take influenza virus H3N2 as example):
(1) at first for the object of monitoring (H3N2), biochip is carried out specific antibody (H3N2 Antibody of Influenza) modify;
(2) startup 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 occur in the electroconductibility of silicon nanowires, far away higher than the electroconductibility of the correspondence of silicon nanowires itself or negative air sample, 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 completed by 4 steps: the first step, under room temperature, nano-device and glutaraldehyde (5% glutaraldehyde, pH=8) reaction is 1 hour, then uses phosphate buffered saline buffer (10mM, pH=8) to rinse 5 minutes.Second step, H3N2 antibody (0.1mg/ml antibody-solutions, pH=8 comprise 4mM sodiumcyanoborohydride) is coated with 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, rinsed 5 minutes with phosphate buffered saline buffer (10mM, pH=8).In the 4th step, utilize Tri N-Propyl Amine solution (100mM Tri N-Propyl Amine, pH=8) coated nano-device, time 2 h.Utilize the amino group sealing nano-device surface of Tri N-Propyl Amine there is no aldehyde groups with antibodies.After corresponding microorganism occurring in air, be sampled the device collection condensing, enter the chip detection part, Ag-Ab immunity association reaction occurs, the bio signal of appearance is converted to electrical signal through preposition amplification and phase-locked amplification, has namely realized on-line monitoring.
Fig. 2 is the partial data of on-line monitoring influenza virus (H3N2 hypotype), according to the information in figure, when having influenza virus (H3N2 hypotype) in air, whole system can show that in the 1-3 minutes the significant electricity of silicon nanowires leads variation, meanwhile, show according to the result of gene amplification, the sample correspondence that in air, influenza virus concentration is high 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 considerably beyond other conventional art, such as gene amplification usually needs 2-3 hour.
To sum up, the present invention has following advantage:
(1) integrated by to technology such as silicon nano-wire biological sensor, micro-fluidic, air continuous sampling and signal locking amplifications of the present invention, formed the bioaerosol real-time monitoring system, result of study shows that when having influenza virus to exist in air, this system can show early warning signal in 1-3 minute;
(2) this system integration the technology of different field comprise 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 air and quantitatively.
Claims (8)
1. the system of a Real-Time Monitoring bioaerosol, is characterized in that, comprises three parts:
-sample collecting and supply unit comprise sampling thief (1), sample delivery pipe (2), peristaltic pump (9), sampling media container (11), collection containers (10); Described sampling thief (1) is realized the collection to bioaerosol, then sampling media is completed the conveying of sample by sample delivery pipe (2) under the effect of peristaltic pump (9), and through micro-fluidic (4), biochip (3), be transported at last collection containers (10);
-signal detection and processing unit, comprise prime amplifier (6), lock-in amplifier (7), be integrated with the described biochip (3) of silicon nano-wire biological sensor (5), biochip (3) is connected with described prime amplifier (6) and described lock-in amplifier (7), and described prime amplifier (6) also is connected with described lock-in amplifier (7);
The output of-data and display unit comprise the host computer and the indicating meter that are connected with lock-in amplifier (7); Be equipped with on described host computer for the data that lock is put and transmit the software that control, signal post-processing and result show;
Wherein: described sampling thief (1) is the electrostatic field sampling thief, comprising: dome electrode (101), circular electrode (102), sample collecting groove (104); Wherein, dome electrode (101) is fixed on insulator foot (106), circular electrode (102) is positioned at the sphere center position of first ball shaped electrode of insulator foot (106) (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) top; Be provided with two airouts (110) on insulator foot (106); Sample delivery outlet (108) and a collection liquid input aperture (109) are arranged in sample collecting groove (104);
In described signal detection and processing unit, the microelectrode of metal probe contact biochip (3) connects input electrical signal by the metal probe lead-in wire and is applied on chip electrode, draws simultaneously detected signal and inputs to surveying instrument; Wherein, two metal probes connect respectively the source-drain electrode of silicon nano-wire biological sensor, be connected with cable between metal probe and lock-in amplifier (7) and prime amplifier (6), a metal probe is connected with the reference output of lock-in amplifier (7), and another metal probe is connected to the voltage input of lock-in amplifier (7) through prime amplifier (6).
2. the system as claimed in claim 1, is characterized in that, the electrode surface material of described biochip (3) is Au, and metal probe needle point material is the composition metal of Au.
3. the system as claimed in claim 1, is characterized in that, the needle point radius-of-curvature of described metal probe needle point is 25 μ m.
4. the system as claimed in claim 1, it is characterized in that, in described sample collecting and supply unit, the pump head of peristaltic pump (9) is two-tube output, one end of one of them flexible pipe connects sampling media, and the other end connects sample collecting groove (104) input interface of sampling thief (1); One end of another flexible pipe connects sample collecting groove (104) output interface, and the other end is connected in the outlet of micro-fluidic (4) on biochip (3).
5. the system as claimed in claim 1, is characterized in that, in described data output and display unit, adopts extendible GPIB card and private cable to connect lock-in amplifier (7) and host computer; Cable is connected with the RS232 serial ports of lock-in amplifier (7); On 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 rear measured electricity that converts is led change curve, and can preserve the data of real-time detection.
6. system as claimed in claim 5, is characterized in that, described computer software has the function of trigger alarm when thing to be checked being detected.
7. a method of utilizing system claimed in claim 1 to carry out the Real-Time Monitoring bioaerosol, is characterized in that, comprises the steps:
A) for the object microorganism A that monitors, biochip (3) is carried out specific antibody modify;
B) startup 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, the electroconductibility that demonstrates silicon nanowires in indicating system occurs significantly to change, far away higher than the electroconductibility of the correspondence of silicon nanowires itself or negative air sample, based on the specificity of antibody, the phenomenon that electric conductivity value significantly changes can confirm the existence of detected object microorganism A.
8. method as claimed in claim 7, is characterized in that, after microorganism A existence being detected, reported to the police by launch computer.
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| CN106525733B (en) * | 2016-10-25 | 2019-04-12 | 西安交通大学 | A kind of microbes in air aerosol device for fast detecting and method |
| US11041833B2 (en) * | 2016-10-26 | 2021-06-22 | Shimadzu Corporation | Flow-through vial and automatic sampler |
| CN108362754B (en) * | 2018-01-19 | 2020-10-09 | 北京大学 | Online detection system and method for biomarkers in exhaled breath |
| CN111529225A (en) * | 2020-06-12 | 2020-08-14 | 吉林大学 | Ambulance with function of immediately detecting new coronavirus in air |
| CN111763614B (en) * | 2020-07-24 | 2023-06-20 | 北京大学 | On-line bioaerosol monitoring system and method based on ATP biochemical luminescence |
| CN112649412A (en) * | 2020-12-29 | 2021-04-13 | 北京鼎蓝科技有限公司 | Bio-aerosol early warning device based on ATP bioluminescence technology |
| CN113295653A (en) * | 2021-05-20 | 2021-08-24 | 北京航空航天大学 | Real-time detection system and method for aerogel pathogens |
| CN114594208A (en) * | 2022-03-07 | 2022-06-07 | 谱瑞前海(深圳)智能科技有限公司 | Real-time detection device for virus spread to epidemic disease in air environment |
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| CN1460855A (en) * | 2003-06-13 | 2003-12-10 | 中国科学院生物物理研究所 | Microfluid biological sensor chip device and its application |
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