CN109298061B - Portable micro cancer antigen multi-parameter quantitative sensing detection system and method - Google Patents
Portable micro cancer antigen multi-parameter quantitative sensing detection system and method Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a portable trace cancer antigen multi-parameter quantitative sensing detection system and a method, comprising the following steps: the micro-fluidic paper chip immunosensor and the sensing rear end signal processing module are electrically connected with each other; the microfluidic paper chip immunosensor comprises an annular filtering membrane, a sample inlet hole and a detection area which are sequentially arranged from top to bottom; the periphery of the sample inlet hole is connected with a plurality of microfluidic channels, and a test sample enters the microfluidic channels after passing through the annular filter membrane, flows into the sample inlet hole from the microfluidic channels and enters the detection area through the sample inlet hole; the detection area adopts a three-electrode system, wherein the working electrode and the counter electrode adopt conductive carbon paste, and the reference electrode adopts silver/silver chloride paste. The high-sensitivity quantitative detection of different cancer protein antigens can be carried out through the microfluidic sensor chip, and the high-sensitivity quantitative detection kit has the scene universal application value of different cancer proteins or polypeptide molecules.
Description
Technical Field
The invention relates to the technical field of micro cancer biosensing detection integration, in particular to a portable micro cancer antigen multi-parameter quantitative sensing detection system and a method.
Background
The lung cancer is one of common malignant tumors, and the early detection, diagnosis and treatment of the lung cancer have great practical value. With the detection of tumor protein markers and polypeptide cancer markers, it is desirable to provide a wearable application that meets the current objectives of transplantation to a terminal device.
The existing biosensing detection integrated system can not meet the requirements of equipment miniaturization, high device integration, system portability, chemistry and intelligent detection.
The traditional electrochemical immunoassay method for the tumor marker has the advantages of less serum detection amount, short analysis time, low cost, high accuracy and precision, simple and convenient operation and good repeatability, and is very suitable for rapid detection on site. However, the conventional tumor marker is mainly detected by a large electrochemical workstation for electrochemical immunodetection, and the instrument has high detection precision, but has high cost, large volume, inconvenience in carrying and inconvenience in integrating into a miniature instrument.
Traditional methods utilize electrochemical immunoassay methods using commercially available chips from different manufacturers, such as: the small signal amplifier, the ADC, the signal processor and the like are integrated on a PCB, and the purpose of single-chip integration of a signal processing part after sensing cannot be met. Therefore, a highly integrated sensing back-end microchip system is urgently needed.
The existing technology utilizes a capacitance biosensor and a large-volume sensing rear-end signal processing system to quickly and conveniently detect the early cancerated cells, but the existing technology does not have the capacity biosensor and the large-volume sensing rear-end signal processing system
In the biological recognition system of the integrated biological sensing chip in the prior art, the MEMS device substrate is silicon, glass and the like, the cost is high, and the sensing rear end detection device has low integration degree and larger volume.
Therefore, the prior art cannot solve the problems of high-sensitivity specific detection of multi-parameter quantitative cancer marker protein and polypeptide molecules, high integration of a single chip of a sensing back-end circuit system and the like.
Disclosure of Invention
In order to solve the problems, the invention provides a portable micro cancer antigen multi-parameter quantitative sensing detection system and a method, the system can be used for high-sensitivity specific wearable application of cancer protein, and the whole system has a multi-parameter detection platform and has the advantages of high integration, high reliability, small volume, low power consumption, long standby time and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one or more embodiments, a portable micro-cancer antigen multi-parameter quantitative sensing detection system is disclosed, comprising: the micro-fluidic paper chip immunosensor and the sensing rear end signal processing module are electrically connected with each other;
the microfluidic paper chip immunosensor comprises an annular filtering membrane, a sample inlet hole and a detection area which are sequentially arranged from top to bottom; the periphery of the sample inlet hole is connected with a plurality of microfluidic channels, and a test sample enters the microfluidic channels after passing through the annular filter membrane, flows into the sample inlet hole from the microfluidic channels and enters the detection area through the sample inlet hole;
the single chip integration of the sensing rear end signal processing module provides DPV detection voltage for the detection of the microfluidic paper chip immunosensor, and the concentration of the tumor marker is automatically calculated according to an electrochemical current signal generated on the microfluidic paper chip immunosensor.
Further, modifying the gold nano/thionine/graphene nano composite film on the working electrode; different antibodies are fixed on the working electrode through the action of the gold nano-particles and the amino groups of the protein.
Further, the sensing back-end signal processing module comprises: the device comprises a processor, a digital-to-analog conversion circuit and a DPV working waveform generation circuit which are connected in sequence; the working electrode is respectively connected with the DPV working waveform generating circuit and the current/voltage conversion circuit, the current/voltage conversion circuit is connected with the analog-to-digital conversion circuit, and the analog-to-digital conversion circuit is connected with the processor.
Further, the processor generates digital DPV detection voltage, the digital-to-analog conversion circuit converts the digital DPV detection voltage into an analog signal and sends the analog signal to the DPV working waveform generation circuit, the DPV working waveform generation circuit applies the analog detection voltage signal to each electrode end of the microfluidic paper chip immunosensor, and an electrochemical current signal is generated on the microfluidic paper chip immunosensor under the action of the voltage; the electrochemical current signal is converted into a voltage signal through a current/voltage conversion circuit, the voltage signal is sent into a processor after passing through an analog-to-digital conversion circuit, and the processor automatically calculates the concentration of the tumor marker according to a calibrated correction equation between the current and the concentration.
Further, the DPV working waveform generating circuit utilizes a transconductance operational amplifier designed as a voltage follower to provide an electrochemical detection reference electrode with a working potential that is not affected by electrochemical current, and this voltage is controlled only by the processor.
Further, the output impedance of a working electrode in the microfluidic paper chip immunosensor is reduced, and the input impedance of the current/voltage conversion circuit is improved, so that the weak current conversion efficiency sensed by the microfluidic paper chip immunosensor is improved.
Further, in the processor, the digital-to-analog conversion circuit and the DPV working waveform generation circuit, based on a noise-first optimization strategy, low-frequency flicker noise, differential input offset noise and out-of-band noise in the DPV working waveform generation circuit are reduced; mismatch noise of the digital-to-analog conversion circuit is reduced, and the processor section is isolated separately from the digital signal section in the digital-to-analog conversion circuit to reduce noise coupling of the digital section to the analog section.
In one or more embodiments, disclosed is a portable microscale cancer antigen multi-parameter quantitative sensing detection method, comprising:
filtering a test sample, and then entering a sample detection area of the microfluidic paper chip immunosensor;
the antibody in the sample detection area is combined with the antigen of the test sample in a high specificity manner to generate electric field change, so that the diffusion of an electroactive substance to the surface of the electrode is hindered, the detected current is reduced, and the concentration of the antigen to be detected and the size of the detected current are in an inverse relation;
calibrating the relation between the concentration of the antigen to be detected and the magnitude of the detection current;
generating a digital DPV detection voltage signal, and applying an analog voltage to each electrode end of the microfluidic paper chip immunosensor after the digital DPV detection voltage signal is subjected to digital-to-analog conversion;
generating an electrochemical current signal on the microfluidic paper chip immunosensor under the action of voltage;
and converting the electrochemical current signal into a voltage signal, and automatically calculating the concentration of the tumor marker according to a calibrated correction equation between the current and the concentration.
Compared with the prior art, the invention has the beneficial effects that:
(1) the sensor integrates a sensing rear end signal processing circuit on an IC chip by using a paper immune sensing micro-fluidic technology, and the integrated detection system has small volume and mass and is convenient to use in a wearing scene.
(2) The high-sensitivity quantitative detection of different cancer protein antigens can be carried out through the microfluidic sensor chip, and the high-sensitivity quantitative detection kit has the scene universal application value of different cancer proteins or polypeptide molecules.
(3) The back-end processing integrated single-chip system of the micro-fluidic paper chip immunosensor can realize the integrated amplification, conversion, quantification, algorithm conversion and correction of a weak electric signal chip, and the power consumption, noise and system reliability are optimized.
(4) Some interference macromolecules are filtered by the annular filtering membrane, and then uniformly reach the position of the sample inlet hole through the multi-branch microfluidic channel, so that the interference macromolecules enter the detection area, interference factors can be fully filtered, and the detection precision is improved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1 is a schematic diagram of a portable micro-cancer antigen multi-parameter quantitative sensing detection system;
FIG. 2(a) is a schematic structural diagram of a microfluidic paper chip immunosensor;
FIG. 2(b) is a schematic structural diagram of a three-electrode system of the microfluidic paper chip immunosensor;
FIG. 3 is a process flow diagram of a sensing backend signal processing module;
FIG. 4 is a DPV voltage waveform generation circuit diagram;
fig. 5 is a current/voltage conversion circuit diagram.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In one or more embodiments, a portable micro-cancer antigen multi-parameter quantitative sensing detection system is disclosed, as shown in fig. 1, comprising: the micro-fluidic paper chip immunosensor and the sensing rear end signal processing module are electrically connected with each other;
the structure of the microfluidic paper chip immunosensor is shown in fig. 2 and comprises an annular filtering membrane, a sample inlet and a detection area which are sequentially arranged from top to bottom; the periphery of the sample inlet hole is connected with a plurality of microfluidic channels, and a test sample enters the microfluidic channels after passing through the annular filter membrane, flows into the sample inlet hole from the microfluidic channels and enters the detection area through the sample inlet hole;
the detection area adopts a three-electrode system, wherein the working electrode and the counter electrode adopt conductive carbon paste, and the reference electrode adopts silver/silver chloride paste.
The three-electrode system comprises two working loops: (1) a voltage polarization loop (which is composed of a working electrode and a reference electrode and realizes control or monitoring of polarization potential of electrochemical reaction, wherein the polarization potential is caused by chemical reaction of a sensitive membrane material on the working electrode;
(2) and a current polarization loop (which is composed of a working electrode and a counter electrode, namely the measurement of the polarization current of the electrochemical reaction of the working electrode is realized, and electrons are transmitted to form the current loop so as to reflect the polarization curve of the chemical reaction on the working electrode).
The working process of the three-electrode system is as follows: firstly, a stable constant voltage is applied to a reference electrode of a voltage polarization loop, a variable potential is applied to a working electrode through a DPV differential pulse voltammetry method, a redox chemical reaction occurs on the surface of a nano composite material sensitive membrane on the working electrode, and a generated weak small current is output through a counter electrode of the current polarization loop.
As shown in fig. 2(a) and 2(b), the serum is dropped on the hydrophilic region of the first layer of the "filter layer" on the carbon paper substrate, which is responsible for filtering and removing the impurity macromolecules, then the serum reaches the "sample region" of the second layer on the carbon paper substrate through the "microfluidic sample-introducing channel" of the first layer, and then reaches the surface reaction region of the "working electrode" on the bottommost layer (back surface) of the carbon paper substrate through the "outlet sample-introducing channel" of the second layer (and the surface of the "working electrode" is pretreated, and the nanocomposite is modified on the surface and a specific selective antibody is fixed on the surface); the 'reference electrode' and the 'counter electrode' with larger areas relative to the working electrode are both positioned on the topmost layer (the first layer) on the carbon paper, a constant voltage-stabilizing potential is applied to the 'reference electrode', the potential difference of a voltage polarization loop formed by the 'working electrode' and the 'reference electrode' is controlled by a DPV differential pulse voltammetry method, then the working electrode performs a chemical oxidation-reduction reaction, the generated weak current reflects a polarization curve reflected by the 'working electrode', and the weak current is transmitted out through the 'counter electrode' of the current polarization loop formed by the 'working electrode' and the 'counter electrode'.
The microfluidic paper chip immunosensor is characterized in that a corresponding hydrophilic area and a corresponding hydrophobic area are manufactured on the basis of a paper base by using a wax-spraying printing technology, wherein the hydrophilic area has strong affinity to water and can be used as a filtering membrane, a microfluidic channel, a sample inlet and a detection area; the hydrophobic area is printed by wax spraying, the positions outside the filtering membrane, the microfluidic channel, the sampling hole and the detection area are all the hydrophobic areas, and a sample cannot enter the hydrophobic area.
Printing on the surface of the chromatographic paper by using a screen printing process to obtain a three-electrode system (a working electrode, a counter electrode and a reference electrode), wherein the working electrode and the counter electrode adopt conductive carbon paste, and the reference electrode adopts Ag/AgCl;
the graphene/thionine/gold nano composite membrane is modified on the surface of the working electrode to fix an antibody, and the antibody is combined with an antigen of a detected object at high specificity to generate electric field change, so that the diffusion of an electroactive substance to the surface of the electrode is hindered, the detected current is reduced, and the concentration of the antigen to be detected and the size of the detected current are in an inverse relation.
The thionine is an electroactive substance, and the current response measured by electrochemical detection is the current change generated by the interaction of the thionine and the surface of the electrode; the concentration of the tumor protein marker can be correspondingly calculated through the direct proportional relation between the reaction current and the concentration.
The carbon paper pulp used as the working electrode is modified with a gold nano/thionine/graphene nano composite membrane, the serum to be detected is dripped on the position of the annular filter membrane, some interfering macromolecules are filtered, and then the serum uniformly reaches the position of the sample injection hole through the multi-branch microfluidic channel, namely enters the detection area.
Through physical adsorption and chemical cross-linking, an electronic mediator, a nano composite material and different antibodies are fixed on the surface of a working electrode of the sensor, so that specific capture and label-free detection of various markers are realized, biological signals are amplified, and meanwhile, the detection sensitivity and the reaction speed can be improved by the composite nano material.
The sensing back-end signal processing module (i.e. the sensing back-end IC chip) is based on the DPV (differential pulse voltammetry) detection principle,
the DPV detection working waveform can be regarded as superposition of linearly increased voltage and constant-amplitude rectangular pulse, the height delta E of the rectangular pulse is a fixed value, and the typical value is 50 mV; the period of the rectangular pulse is generally 500-2000 ms; the pulse width is generally 40-80 ms. The current values were measured twice in one cycle: measuring the current respectively 20ms before the pulse and 20ms after the pulse, subtracting the two measured currents to obtain a difference current delta i as the current value at the moment, continuously measuring the current delta i in a plurality of periods, and drawing a relation graph between the delta i and the potential E, namely a differential pulse curve.
Wherein Δ i satisfies:
Δi＝(n 2*F 2)/4*R*T*A*ΔE*D 1/2(π*tm)-1/2*C (1)
in the above formula (1), Δ i is proportional to the substance concentration C, which is the basis of quantitative analysis;
the measured current is the sum of electrolytic current and capacitance current 20ms after the pulse period, the current is subtracted twice to obtain delta i, the interference of the capacitance current is reduced, and the background current caused by the oxidation reduction current of impurities is greatly offset, so that the DPV detection method adopted by the rear end of the sensor has higher detection sensitivity and accuracy than the conventional chronoamperometry and cyclic voltammetry scanning method.
As shown in fig. 1, the sensing back-end signal processing module includes: the device comprises a processor, a display, a digital-to-analog conversion circuit (DAC) and a DPV working waveform generating circuit which are connected in sequence; the working electrode is connected with the DPV working waveform generating circuit and the current/voltage converting circuit respectively, the current/voltage converting circuit is connected with the analog-to-digital converting circuit (ADC), and the analog-to-digital converting circuit and the display are connected with the processor respectively.
The processor controls the generation of an adjustable digital DPV voltage, which is then converted to an analog DPV voltage using a DAC, which provides a stable lateral potential voltage for the working electrode.
The DPV working waveform generating circuit is designed into a voltage follower by using a transconductance operational amplifier as shown in FIG. 4 to provide an electrochemical detection reference electrode with a working potential which is not influenced by electrochemical current, and the voltage is only controlled by a processor; the output voltage Vout of the DPV voltage waveform generation circuit is equal to the input voltage Vin (output follow input), Vdd > Vout > Vss, and Vout provides stable working voltage for the working electrode of the sensor.
As shown in fig. 5, the current-voltage conversion circuit converts the measured current signal into a voltage signal that can be easily measured, and sends the voltage signal to the ADC module for subsequent data calculation. In fig. 5, a current Ii flows through a resistor R1, a voltage is generated across a resistor R1, and an amplifier differentially amplifies the voltage across R1; the adjustable resistor connected with V1 is used for zero adjustment (eliminating the influence of input mismatch) to eliminate the zero point error of the circuit; the other adjustable resistor is used for adjusting the amplification factor.
The output impedance of a working electrode of the sensor is reduced, and the input impedance of a current-voltage conversion circuit at the rear end of the sensor is improved, so that the weak current conversion efficiency sensed by the sensor is improved; in addition, in the design of a processor, a DAC and a voltage follower generating a lateral potential, based on a strategy of noise optimization priority, low-frequency flicker noise, differential input offset noise, out-of-band noise, mismatch noise of a DAC module and the like in a circuit of the voltage follower generating the lateral potential are sufficiently reduced, and a digital CPU part is separately isolated from a digital part in an analog module so as to reduce noise coupling of digital logic to the analog part circuit.
The working process of the sensing rear-end signal processing module is shown in fig. 3, and specifically includes:
the processor generates a digital DPV detection voltage, and converts the digital DPV (differential-pulse-voltage) detection voltage into an analog signal through an internal DAC (digital-to-analog converter) module of the sensing back-end IC chip and sends the analog signal to a DPV (differential-pulse-voltage) working waveform generating circuit;
the DPV working waveform generating circuit applies detection voltage to each electrode end of the microfluidic paper chip, and weak electrochemical current signals are generated on the microfluidic paper chip under the action of the voltage; the weak current is converted into a voltage signal through a current-voltage conversion circuit; the voltage signal obtained by conversion is sent to a processor and is converted into a digital signal through an internal ADC (analog to digital converter) at the rear end of the sensor; the concentration of the tumor marker is in proportional relation with the detected electrochemical current signal, and the IC chip at the rear end of the sensor can automatically calculate the concentration of the tumor marker according to a calibrated correction equation between the current and the concentration.
The electrochemical detection object is the tumor marker concentration (mu g/L), the detection is carried out according to the principle that the specific binding of antigens with different concentrations and antibodies can cause the electrochemical current response change of electroactive substances, the current response is very weak (nA-mu A level), and therefore the weak current signal can be detected only by converting the weak current signal into a voltage signal which is easy to measure.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the present invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive efforts by those skilled in the art based on the technical solution of the present invention.
Claims (5)
1. A portable micro cancer antigen multi-parameter quantitative sensing detection system is characterized by comprising: the micro-fluidic paper chip immunosensor and the sensing rear end signal processing module are electrically connected with each other;
the microfluidic paper chip immunosensor comprises an annular filtering membrane, a sample inlet hole and a detection area which are sequentially arranged from top to bottom; the periphery of the sample inlet hole is connected with a plurality of microfluidic channels, and a test sample enters the microfluidic channels after passing through the annular filter membrane, flows into the sample inlet hole from the microfluidic channels and enters the detection area through the sample inlet hole;
the single chip integration of the sensing rear end signal processing module provides DPV detection voltage for the detection of the microfluidic paper chip immunosensor, and the concentration of the tumor marker is automatically calculated according to an electrochemical current signal generated on the microfluidic paper chip immunosensor;
the sensing back end signal processing module comprises: the device comprises a processor, a digital-to-analog conversion circuit and a DPV working waveform generation circuit which are connected in sequence; the working electrode is respectively connected with the DPV working waveform generating circuit and the current/voltage conversion circuit, the current/voltage conversion circuit is connected with the analog-to-digital conversion circuit, and the analog-to-digital conversion circuit is connected with the processor;
the output impedance of a working electrode in the microfluidic paper chip immunosensor is reduced, and the input impedance of a current/voltage conversion circuit is improved, so that the weak current conversion efficiency sensed by the microfluidic paper chip immunosensor is improved;
in a processor, a digital-to-analog conversion circuit and a DPV working waveform generating circuit, based on a noise-first optimization strategy, low-frequency flicker noise, differential input offset noise and out-of-band noise in the DPV working waveform generating circuit are reduced; mismatch noise of the digital-to-analog conversion circuit is reduced, and the processor part is isolated from the digital signal part in the digital-to-analog conversion circuit separately, so that noise coupling of the digital part to the analog part is reduced;
the system is used for non-disease diagnostic treatment.
2. The portable microscale cancer-antigen multi-parameter quantitative-sensing detection system of claim 1, wherein a gold nano/thionine/graphene nano composite membrane is modified on the working electrode; different antibodies are fixed on the working electrode through the action of the gold nano-particles and the amino groups of the protein.
3. The portable micro-cancer antigen multi-parameter quantitative sensing detection system as claimed in claim 1, wherein the processor generates digital DPV detection voltage, the digital-to-analog conversion circuit converts the digital DPV detection voltage into analog signals and sends the analog signals to the DPV working waveform generation circuit, the DPV working waveform generation circuit applies the analog detection voltage signals to each electrode terminal of the microfluidic paper chip immunosensor, and electrochemical current signals are generated on the microfluidic paper chip immunosensor under the action of the voltage; the electrochemical current signal is converted into a voltage signal through a current/voltage conversion circuit, the voltage signal is sent into a processor after passing through an analog-to-digital conversion circuit, and the processor automatically calculates the concentration of the tumor marker according to a calibrated correction equation between the current and the concentration.
4. The portable micro-cancer antigen multi-parameter quantitative sensing detection system of claim 1, wherein the DPV working waveform generation circuit utilizes a transconductance operational amplifier designed as a voltage follower to provide an electrochemical detection reference electrode with an operating potential that is not affected by electrochemical current, and the voltage is controlled only by the processor.
5. A detection method using the portable micro cancer antigen multi-parameter quantitative sensing detection system of any one of claims 1 to 4, comprising:
filtering a test sample, and then entering a sample detection area of the microfluidic paper chip immunosensor;
the antibody in the sample detection area is combined with the antigen of the test sample in a high specificity manner to generate electric field change, so that the diffusion of an electroactive substance to the surface of the electrode is hindered, the detected current is reduced, and the concentration of the antigen to be detected and the size of the detected current are in an inverse relation;
calibrating the relation between the concentration of the antigen to be detected and the magnitude of the detection current;
generating a digital DPV detection voltage signal, and applying an analog voltage to each electrode end of the microfluidic paper chip immunosensor after the digital DPV detection voltage signal is subjected to digital-to-analog conversion;
generating an electrochemical current signal on the microfluidic paper chip immunosensor under the action of voltage;
converting the electrochemical current signal into a voltage signal, and automatically calculating the concentration of the tumor marker according to a calibrated correction equation between the current and the concentration;
when the electrochemical current signal is converted into a voltage signal, the output impedance of a working electrode in the microfluidic paper chip immunosensor is reduced, the input impedance of a current/voltage conversion circuit is improved, and the weak current conversion efficiency sensed by the microfluidic paper chip immunosensor is improved;
in a processor, a digital-to-analog conversion circuit and a DPV working waveform generating circuit, based on a noise-first optimization strategy, low-frequency flicker noise, differential input offset noise and out-of-band noise in the DPV working waveform generating circuit are reduced; mismatch noise of the digital-to-analog conversion circuit is reduced, and the processor section is isolated separately from the digital signal section in the digital-to-analog conversion circuit to reduce noise coupling of the digital section to the analog section.
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