CN1996001A - Biosensor for monitoring biological behavior of cell and biomolecule and sensing method therefor - Google Patents

Abstract

The biological sensor is particularly used for monitoring the behavior of the cells, nucleic acid and protein against the resistance technique. There are two or more electrodes attached to the insulation base which enjoys good biological compatibility with the electrode material. Through resistance to the measurement sensor, it can realize direct, timely inspection for the behavior of the cells and big molecules. It has automatic and timely monitoring for the cells with sensitivity, timely, continuous features, free from chemical mark and chemical poison to the cells, easy realization of automatic measurement.

Description

The biology sensor and the method for sensing of monitoring cell and biomolecule biological behaviour

Technical field

The present invention relates to field of biosensors, particularly monitor based on the biological behaviour of resistance technique pair cell and biomolecule such as nucleic acid, albumen.The present invention specifically comprises device and the method for sensing that utilizes resistance technique monitoring cell and biomacromolecule biological behaviour.

Background technology

The traditional monitoring cell and the method for biomacromolecule biological behaviour have a lot, but mainly are some biochemical and immunologic methods, for example use fluorochrome label, use ultraviolet excitation then, detect with fluorescent microscope.With MTT/XTT colorimetric method for determining cell competence for added value is a representative illustration measuring the cell biology behavior with biochemical method, the principle of this method is: in the vigorous normal cell of metabolism, its Intramitochondrial succinate dehydrogenase, tetrazolium salts substances (as MTT, XTT, WST-1 etc.) can be reduced to the crystalloid material of purple, be deposited on cell peripheral, thereby the amount of purple crystal has reflected the size of cell competence for added value.Thereby, as long as read the OD value, just can be measured to the increment state (Mosmann T.Rapid colorimetric assay for cellular growth and survival:application to proliferationand cytotoxicity assays.J Immunol Methods.1983 6:55-63.) of cell by microplate reader.The advantage of MTT method is that mainly its operation is simple relatively, highly sensitive, thereby has obtained more widely and used.But, the problem that this class also exists some to be difficult to overcome with the method for biological chemistry and immunology means monitoring cell biology behavior, for example can not be continuously, monitoring in real time, need carry out chemical labelings such as fluorescence or antibody, pair cell can cause certain chemistry to poison, and is difficult to realize supermatic monitoring or the like.

The method of some biophysicss can overcome in the above-mentioned shortcoming one even multinomial to a certain extent, for example change of the method monitoring cytoactive of electricity consumption rotation and cell biology behavior under drug effect (the apparatus and method application (patent) that the rotation of Chinese patent high-flux electric detects number: 00124086.2 Cheng Jing; Xu Junquan; Zhu Xiaoshan; Liu Litian; Wang Xiaobo; Wu Lei).Based on the cell of impedance measurement and the monitoring method of biomacromolecule biological behaviour also is a kind of very sensitive, can be in real time, continuous coverage, need not the bio-physical method of chemical labeling, relevant (the Giaever I.and Keese C.R. of elaboration is arranged in the documents and materials below, " Monitoring fibroblast behavior in tissue culture with an applied electric field ", Proc.Natl.Acad.Sci. (USA), 1984, vol.81, pp 3761-3764).

The cell electrical impedance is mainly determined by the ionic environment in device top electrode and solution interface and the solution, simultaneously also by the attaching state decision of cell on electrode, for example when cell stretched on electrode, with the contact area increase of electrode, at this moment resistance value just can rise; Otherwise impedance then can reduce in the time of cell death.When the state of cell, when for example the form of cell, fertility, attaching state etc. changed, resistance value also can change accordingly, has had the method for the impedance measurement of experimental results show that more sensitiveer than some biochemical methods commonly used.

Summary of the invention

The object of the present invention is to provide a kind of biology sensor and method for sensing of monitoring cell and biomolecule biological behaviour.This sensor is characterised in that: adhere to two or more electrodes on the matrix of insulation, the matrix of insulation and the material of electrode are the material of good biocompatibility; The electrode of described sensor has two at least; The material of the dielectric substrate of described sensor is to comprise glass or silicon or macromolecule polymer material or their insulating material that is combined in interior good biocompatibility; The electrode of described sensor is the conductive material that comprises in metal or nonmetal or they be combined in.

The monitoring cell that the present invention proposes and the method for sensing of biomolecule biological behaviour, it is characterized in that: this method contains following step successively: the first, cultured cell or stick DNA or the protein biomacromolecule on sensor; The second, sensor and current-limiting resistance polyphone back are inserted measurement mechanism; The 3rd, load the simple alternating current electric signal on the series circuit of measurement mechanism to sensor and resistance formation; The 4th, measurement mechanism is the sinusoidal voltage signal on the survey sensor and the sinusoidal voltage signal of loading simultaneously, and according to the impedance of measurement result calculating sensor , computing formula is as follows:

$\stackrel{.}{Z}=\frac{{\stackrel{.}{U}}_2}{{\stackrel{.}{U}}_1}R$

Wherein Be the simple alternating current electric signal on the sensor, For being carried in the sinusoidal voltage signal at sensor and current-limiting resistance two ends, R is the resistance of current-limiting resistance.

The present invention utilizes this sensor and method for sensing to monitor the THP-1 cell in real time and induces the process that is divided into the THP-1 macrophage down at phorbol ester, experimental provision comprises sensor, impedometer and a computing machine, the frequency of the pumping signal that we adopt in experiment is 1KHz, 20KHz, 30KHz, 40KHz, 50KHz, 80KHz, 100KHz, 200KHz and 250KHz, and the amplitude of voltage is 2V.In experiment, we have monitored the variation of THP-1 cell differentiation overall process middle impedance curve in real time.

This method also has sensitivity, can be in real time, continuous coverage, need not chemical labeling, and can not cause chemistry to poison by pair cell, and easily be automated advantage such as measurement.

Description of drawings

Figure 1A and Figure 1B are the synoptic diagram of cell impedance bioelectrical measurement device, and wherein Figure 1B is an enlarged drawing local among Figure 1A.The 1-electrode, the 2-dielectric substrate.

Fig. 2 is the theory diagram of measurement mechanism.

The schematic diagram of Fig. 3 impedance computation and formula explanation thereof.

Embodiment

Technical scheme of the present invention is to carry out cellular incubation on the microelectrode device that utilizes micro-processing technology to make, simultaneously, faint in addition AC field on electrode, when cell can cause inhibition (being impedance) to electric field adherent growth the time on micro element, when cell owing to growing, rise in value, break up and when receiving that medicine irritation causes its form to change, its inhibition to electric field (being impedance) just can change, thereby, by monitoring to its impedance, biological behaviour that just can the indirect monitoring cell.Equally, when biomacromolecule attached to the microelectrode device on the time, also can bring inhibition to faint AC field, when some biological behaviour of biomolecule changes, for example antigen and antibody generation combination, also can bring the change of impedance, by monitoring to its impedance, biological behaviour that just can the indirect monitoring biomacromolecule.

Device provided by the present invention comprises that attached to the nonisulated electrode on the insulator, insulator can be glass, silicon, high molecular polymer, for example material such as polycarbonate.Electrode can be metals such as gold, also can be metal oxides such as tin indium oxide.On the device two or more electrodes are arranged, and electrode is drawn device, link to each other, measure resistance value with measurement mechanism with lead-in wire.During measurement, need to produce the sine wave signal of certain frequency and amplitude, resistance (K ohm---M ohmage magnitude by polyphone, concrete resistance is determined according to the impedance of device) the access measurement mechanism, the resistance of resistance is about about thousand times of the sensor resistance value, thereby makes working sensor in the constant current source pattern.Data acquisition module is a digital signal with voltage signal on the sensor and total voltage analog signal conversion respectively, collects control module, by software analysis, draws the impedance information of sensor, comprises the mould and the phase place of impedance.This measurement mechanism can provide the impedance information under a plurality of frequencies, and the impedance of measuring under each frequency only needs maximum 5 seconds, that is to say if only measure an impedance under the frequency, can measure once in 5 seconds, has reached real-time Testing requirement.

Used at this, " biological behaviour " comprises but not merely comprise the variation that increment, differentiation and the cell of metamorphosis, the cell of cell are taken place under drug effect; Comprise the interaction of biomacromolecules such as not merely comprising albumen, nucleic acid etc.

The processing of device and encapsulation

Among the present invention, the electrode that is used for impedance measurement be on glass matrix with little processing photoetching process commonly used, carve the figure of electrode, then with vacuum evaporation process with golden evaporation on the glass matrix surface, remove the electrode part that forms sensor behind the photoresist.With ultraviolet glue above-mentioned electrode is bonded in the plastic culture dish bottom, and lead and electrode are bonded together with conducting resinl, conducting resinl solidifies covering ultraviolet glue above the back, prevents the toxicity that pair cell may cause when carrying out cellular incubation, and lead is drawn double dish and linked to each other with measurement mechanism.

Claims (5)

1. monitor the biology sensor of cell and biomolecule biological behaviour, this sensor is characterised in that: adhere to two or more electrodes on the matrix of insulation, the matrix of insulation and the material of electrode are the material of good biocompatibility.

2. the biology sensor of monitoring cell as claimed in claim 1 and biomolecule biological behaviour is characterized in that, the electrode of this sensor has two at least.

3. the biology sensor of monitoring cell as claimed in claim 1 and biomolecule biological behaviour, it is characterized in that the material of the dielectric substrate of this sensor is to comprise glass or silicon or macromolecule polymer material or their insulating material that is combined in interior good biocompatibility.

4. the biology sensor of monitoring cell as claimed in claim 1 and biomolecule biological behaviour is characterized in that, the electrode of this sensor is the conductive material that comprises in metal or nonmetal or they be combined in.

5. the method for sensing of monitoring cell and biomolecule biological behaviour, it is characterized in that: this method contains following step successively: the first, cultured cell or stick DNA or the protein biomacromolecule on sensor; The second, sensor and current-limiting resistance polyphone back are inserted measurement mechanism; The 3rd, load the simple alternating current electric signal on the series circuit of measurement mechanism to sensor and resistance formation; The 4th, measurement mechanism is the sinusoidal voltage signal on the survey sensor and the sinusoidal voltage signal of loading simultaneously, and according to the impedance of measurement result calculating sensor , computing formula is as follows:

$\stackrel{.}{Z}=\frac{{\stackrel{.}{U}}_2}{{\stackrel{.}{U}}_1}R$

Wherein Be the simple alternating current electric signal on the sensor, For being carried in the sinusoidal voltage signal at sensor and current-limiting resistance two ends, R is the resistance of current-limiting resistance.

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