CN112098465B - Microfluidic carbohydrate metabolism analysis detection device and method - Google Patents
Microfluidic carbohydrate metabolism analysis detection device and method Download PDFInfo
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Abstract
The application provides a microfluidic carbohydrate metabolism analysis detection device, which comprises: the micro-fluidic device comprises a micro-fluidic sensor unit, a micro-injection pump, a waste liquid pool, a signal circuit, an excitation light source, a light source modulation circuit and a controller; the liquid inlet channel of the microfluidic sensor unit is communicated with the microinjection pump, and the liquid outlet channel of the microfluidic sensor unit is communicated with the waste liquid pool; the signal circuit is connected with the microfluidic sensor unit, applies bias voltage to the microfluidic sensor unit, collects detection signals from the microfluidic sensor unit, is connected with the light source modulation circuit and sends modulation control signals; the excitation light source is arranged towards the microfluidic sensor unit and connected with the light source modulation circuit, and emits excitation light for generating photocurrent to the microfluidic sensor unit; the light source modulation circuit is connected with the excitation light source and modulates the excitation light according to the modulation control signal; the controller is in communication connection with the signal circuit and is used for detecting, controlling, recording and processing the acquired data. The application can realize quantitative analysis and detection of carbohydrate metabolism dynamics.
Description
Technical Field
The application relates to the field of biosensors, in particular to a microfluidic carbohydrate metabolism analysis detection device and a microfluidic carbohydrate metabolism analysis detection method.
Background
The taste sense is a sense produced by the stimulation of the taste organochemical sensing system by food in the mouth of a person and includes four basic tastes of sweet, sour, bitter and salty. The various tastes that people commonly taste are the result of a mix of these four tastes. The taste receptor is taste bud on tongue, which is oval and mainly composed of taste cells and supporting cells, wherein microvilli are arranged on top of taste cells and extend towards the direction of taste pores, and are contacted with saliva, and nerve fibers are arranged on the cell base. The chemicals in the food dissolve in saliva, contacting the taste receptor cells through the taste pores, where they interact with taste receptors or ion channels. These interactions trigger a signaling cascade within the cell, inducing action potentials in the cell, and electrical signals are ultimately transmitted through nerve fibers to the brain, forming a taste sensation. There are many taste sensing molecules on the surface of taste cells, and different substances can bind to different taste sensing molecules to present different tastes. The human taste only needs 1.5-4.0ms from the stimulation of food to the feeling of taste, which is faster than the visual sense of 13-45ms, the hearing of 1.27-21.5ms and the touch of 2.4-8.9 ms. In the past, the taste analysis is mainly judged by adopting an artificial taste analysis method, has high subjectivity, and is difficult to accurately analyze and evaluate the taste brought by food. Therefore, the on-site, rapid and real-time detection can be realized through the taste sensor, and the sensor has important significance for the food field. In particular, it is necessary to achieve dynamic detection in feeding-induced flow conditions.
Sugar is an important flavoring agent and is classified into high-calorie sweeteners and low-calorie sweeteners according to the calories contained therein. The high calorie sweetener comprises natural sweetener such as sucrose and Mel, and has high calorie, and can easily cause obesity and even diabetes after long-term excessive consumption. Low-calorie sweetener is a substance having a sweet taste, low heat generation and low nutritive value, and is generally used for controlling blood sugar elevation, preventing obesity, controlling weight and preventing cardiovascular diseases, and also used as a sugar substitute for diabetics. Lactic acid bacteria in the oral cavity of humans and other mammals react with trace sugar in food to produce organic acids, including lactic acid, acetic acid (ethanol) and other products, which impart specific tastes to different sugars. The metabolic characteristics of the lactobacillus on different saccharides are accurately analyzed through the biosensor, and the method has great significance for preparing the sweetener which is low in calories and has the taste approaching to sucrose in the food field.
Disclosure of Invention
The application aims at overcoming the defects of the prior art, and provides a microfluidic sugar metabolism analysis and detection device and method, which solve the problems of strong subjectivity, poor accuracy caused by static detection objects and the like of conventional taste analysis and detection in the prior art.
The application provides a microfluidic carbohydrate metabolism analysis detection device, which comprises: the micro-fluidic device comprises a micro-fluidic sensor unit, a micro-injection pump, a waste liquid pool, a signal circuit, an excitation light source, a light source modulation circuit and a controller; the liquid inlet channel of the microfluidic sensor unit is communicated with the microinjection pump, and the liquid outlet channel of the microfluidic sensor unit is communicated with the waste liquid pool; the signal circuit is connected with the microfluidic sensor unit, applies bias voltage to the microfluidic sensor unit and collects detection signals from the microfluidic sensor unit, and is connected with the light source modulation circuit to send modulation control signals; the excitation light source is arranged towards the microfluidic sensor unit and connected with the light source modulation circuit, and emits excitation light for generating photocurrent to the microfluidic sensor unit; the light source modulation circuit is connected with the excitation light source and modulates the excitation light source to emit excitation light according to the modulation control signal; the controller is in communication connection with the signal circuit and is used for detecting, controlling, recording and processing the acquired data.
Preferably, the microfluidic sensor unit comprises a substrate, an optical addressing potential sensor, a microporous membrane, a liquid inlet pipe, a liquid outlet pipe and a working electrode; the conducting layer of the optical addressing potential sensor is connected with the working electrode, and the optical addressing potential sensor and the working electrode are arranged on the substrate; the microporous membrane is arranged on the optical addressing potential sensor and is provided with a spinning vertical micro-launder; the liquid inlet pipe and the liquid outlet pipe are arranged at two ends of the micro-flow groove and are inserted into the micro-porous membrane.
Preferably, the liquid inlet pipe and the liquid outlet pipe are composed of a metal pipe and a polyethylene microporous pipe, the metal pipe is inserted into the microporous membrane, and the polyethylene microporous pipe is sleeved on the metal pipe. The metal tube is made of stainless steel, silver or platinum. The metal tube of the liquid inlet tube or the metal tube of the water outlet tube is used as a reference electrode.
Preferably, the optical addressing potential sensor is sequentially composed of an aluminum layer, a silicon wafer substrate, an insulator layer and a protective layer from the back surface to the front surface; the aluminum layer is connected with the working electrode, the protective layer is positioned below the microporous membrane, and the protective layer exposed by the micro-groove is arranged towards the excitation light source.
Preferably, the signal circuit comprises a microcontroller and a LAPS detection circuit; the LAPS detection circuit comprises an I/V conversion circuit, a zeroing and amplifying circuit, a low-pass filter circuit, an impedance detection circuit and a clock circuit; the photocurrent signal of the optical addressing potential sensor is converted into a voltage signal through an I/V conversion circuit; the voltage signal is subjected to error removal and in-phase amplification through a zeroing and amplifying circuit, low-frequency noise is filtered through a low-pass filter circuit, and the low-frequency noise is sent to the microcontroller; the impedance circuit provides a high-frequency alternating current excitation source for the light source modulation circuit to perform impedance detection and drive the excitation light source; the clock circuit provides a clock signal to the impedance circuit through microprocessor control.
Preferably, a bubble eliminating unit is arranged on the liquid inlet channel, and the bubble eliminating unit is communicated with the vacuum pump. The bubble eliminating unit comprises an upper substrate, a lower substrate, a negative pressure cavity, a fluid cavity, a vacuum pump connecting port, a micro-inflow port, a micro-outflow port, a vacuum pump connecting pipe and a fluid conduit; the negative pressure cavity is communicated with the fluid cavity, the negative pressure cavity and the fluid cavity are positioned between the upper substrate and the lower substrate, the negative pressure cavity is adjacent to the upper substrate, and the fluid cavity is adjacent to the lower substrate; the vacuum pump connecting port is positioned on the upper substrate and communicated with the negative pressure cavity; the micro-inflow port and the micro-outflow port are positioned on the lower substrate and are communicated with the fluid cavity.
A method of analytical detection of carbohydrate metabolism comprising: calibrating the microfluidic sensor unit and determining a bias voltage range; culturing lactobacillus and inoculating into a microfluidic sensor unit; delivering a sugar solution to the microfluidic sensor unit, applying a bias voltage and modulating excitation light; recording the detection signal in real time, and drawing the lactic acid bacteria metabolism curve of the sugar solution according to the detection signal.
Preferably, the bias voltage ranges from-1.5V to-1.0V; the density of lactobacillus is 0.5-3×10 7 CFU/ml; the conveying speed of the sugar solution is 300-600mL/h; the detection temperature is 35-40 ℃.
Preferably, the sugar solution comprises 75mmol/L sucrose solution and mixed solution containing sucralose, erythritol and maltitol and having sweetness consistent with that of 75mmol/L sucrose solution.
Preferably, the sugar solution comprises 75mmol/L sucrose solution and mixed solution containing sucralose, erythritol, maltitol, and vitamin C, having sweetness consistent with that of 75mmol/L sucrose solution and acidity of pH 3.0-4.0.
Compared with the prior art, the embodiment of the application has the beneficial effects that: according to the application, the dynamic quantitative analysis and detection of the carbohydrate lactic acid bacteria metabolism are realized by organically combining the optical addressing potential sensor with the microfluidic sensor unit; by arranging the bubble eliminating unit on the liquid inlet channel, the bubble eliminating unit is communicated with the vacuum pump, so that bubbles in the detection solution are effectively eliminated, and the detection precision is improved; based on the analysis and detection device, the application can accurately grasp the metabolic characteristics of lactic acid bacteria of different sugars, and can accurately obtain the acidity range of the compound sweetener with metabolic taste close to that of sucrose.
Drawings
The above features and advantages of the present application will become more apparent and readily appreciated from the following description of exemplary embodiments thereof, taken in conjunction with the accompanying drawings.
Fig. 1 is a schematic structural diagram of a microfluidic carbohydrate metabolism analysis detection device according to an embodiment of the present application.
Fig. 2 is a schematic structural diagram of a microfluidic sensor unit according to an embodiment of the present application.
Fig. 3 is a schematic diagram of an optical addressing potential sensor according to an embodiment of the present application.
Fig. 4 is a circuit configuration diagram of a signal circuit according to an embodiment of the present application.
FIG. 5 is a schematic diagram of a microfluidic carbohydrate metabolism analysis assay device according to another embodiment of the application.
Fig. 6 is a schematic diagram of a bubble eliminating unit according to an embodiment of the present application.
Fig. 7 is a flow chart of the microfluidic carbohydrate milk metabolic analysis detection method of the present application.
Detailed Description
The present application is described in further detail below with reference to the drawings so as to facilitate the accurate understanding of the present application by those skilled in the art.
Example 1
As shown in fig. 1, the microfluidic carbohydrate metabolism analysis detection device 100 provided in this embodiment includes: a controller 110, a signal circuit 120, an excitation light source 130, a light source modulation circuit 140, a microfluidic sensor unit 150, a microinjection pump 160, and a waste liquid tank 170. The liquid inlet channel of the microfluidic sensor unit 150 is connected with the microinjection pump 160, the liquid outlet channel of the microfluidic sensor unit 150 is connected with the waste liquid pool 170, and the microfluidic sensor unit 150 is connected with the signal circuit 120, so that bias voltage and signal acquisition are applied to the microfluidic sensor unit 150. The excitation light source 130 is disposed above the microfluidic sensor unit 150 and connected to the light source modulation circuit 140, so that the microfluidic sensor unit 150 is excited by light to generate photo-generated current. The light source modulation circuit 140 is connected to the signal circuit 120 and is controlled by the signal circuit 120. The signal circuit 120 is connected to the controller 110 through a serial port, and performs the functions of related control, data transmission and processing.
The micro syringe pump 160 may control the injection amount of the test solution through an asynchronous receive/transmit protocol (UART). The excitation light source 130 is composed of a fixed-focus tunable infrared laser diode module for generating electron hole pairs.
[ microfluidic sensor cell ]
As shown in fig. 2, the microfluidic sensor unit 150 of the present embodiment includes a substrate 151, an optical addressing potential sensor 152, a microporous membrane 153, a liquid inlet pipe 154, a liquid outlet pipe 155, and a working electrode 156. The substrate 151 is a PMMA plate of 0.5-1 mm. The back surface of the optical addressing potential sensor 152 has a conductive layer connected to the working electrode 156, and the back surface of the optical addressing potential sensor 152 and the working electrode 156 are fixed on the substrate 151 by conductive adhesive. The microporous membrane 153 is fixed on the front surface of the optical addressing potential sensor 152 by a biological characteristic stable adhesive, the microporous membrane 153 is provided with a spinning vertical micro-flow groove, and the liquid inlet pipe 154 and the liquid outlet pipe 155 are arranged at two ends of the micro-flow groove, inserted into and fixed with the microporous membrane 153. The preparation process flow of the microfluidic sensor unit 150 comprises:
step S210: selecting a PMMA plate with the thickness of 1mm, cleaning and drying, and then cutting into a spinned vertical PMMA plate with the length of 10mm and the short diameter of 3.6mm by using laser, wherein the spinned vertical PMMA plate is used as a die of a micro-flow cavity;
step S220: attaching the spun vertical PMMA sheet to the bottom of the cleaned and dried culture dish by using a double-sided adhesive tape;
step S230: injecting PDMS in a culture dish, uniformly mixing PDMS and a curing agent according to the proportion of 10:1, standing in a vacuum chamber for 20 minutes to remove bubbles, casting into the culture dish, and drying at 80 ℃ for 2 hours to form a PDMS film of 2-3mm for preparing a microporous film;
step S240: taking out the formed PDMS film, and cutting the PDMS film along the edge of the spindly vertical PMMA sheet contained in the PDMS film by using a cutter to form a spindly vertical micro-flow cavity;
step S250: cutting the PDMS film into a square with the length of 15mm multiplied by 15mm, and aligning the center of the microfluidic cavity with the center of the square;
step S260: drilling holes on the PDMS film at the positions corresponding to the tops of the spinning vertical micro-grooves to form an inlet and an outlet, wherein the diameters of the holes are 300-500 mu m;
step S270: attaching a square PDMS film to the front surface of an optical addressing potential sensor with the same size by using a biological characteristic stable adhesive;
step S280: metal tubes in interference fit with the holes are inserted into the inlet and the outlet of the PDMS film, the metal tubes are made of high-stability metal (such as stainless steel, pt and the like), polyethylene microporous tubes are sleeved on the metal tubes and respectively used as a liquid inlet tube and a liquid outlet tube, and meanwhile, the metal tubes are used as a reference electrode and an auxiliary electrode;
step S290: leads and working electrodes are led out from the back of the optical addressing potential sensor and are fixed to a cleaned and dried substrate, for example a 1mm thick PMMA plate, with conductive glue.
Optical addressing potential sensor
As shown in fig. 3, the optical addressing potential sensor 152 of the present embodiment is composed of an aluminum layer 31, a silicon wafer substrate 32, an insulator layer 33, and a protective layer 34 in this order from the back surface to the front surface. The specific processing flow is as follows:
step S310: selecting an n-type single-sided polished silicon wafer with the thickness of 500 mu m, cutting the silicon wafer into square silicon single pieces with the thickness of 15mm multiplied by 15mm, cleaning the square silicon single pieces by an RCA standard cleaning process, and drying the square silicon single pieces;
photoetching and thinning the LAPS corresponding area on the back of the silicon single chip by using a 5% hydrofluoric acid wet chemical etching method to form a silicon chip substrate 32 so as to improve the intensity of response current in the process of photoexcitation;
step S320: by thermal dry oxidation (temperature)The temperature is 1000 ℃ and the time is 40 min) a layer of SiO of 30nm is grown on the corresponding area of the LAPS on the front surface of the silicon substrate 32 2 Forming an insulator layer 33;
step S330: siO deposition by Plasma Enhanced Chemical Vapor Deposition (PECVD) 2 Forming a 50nm silicon nitride layer on the surface as a protective layer;
step S340: and forming an aluminum layer with the thickness of 300nm in a LAPS corresponding area on the back surface of the silicon wafer substrate by thermal evaporation deposition to serve as a conductive layer.
The reference electrode is connected to the signal circuit 120 and cooperates with the auxiliary electrode to provide a dc bias voltage to the optical addressing potential sensor 152 to form a space charge region at the insulator/semiconductor interface. The excitation light source 130 irradiates the silicon wafer substrate 32 to generate electron-hole pairs, and the electron-hole pairs are subjected to the electric field of the space charge region to form photocurrent, and the photocurrent is influenced by the surface potential and has alternating amplitude. The optical addressing potential sensor 152 is in direct contact with the analysis solution, so that the surface potential changes, the intensity of photocurrent is changed, and quantitative analysis of substances is realized. The aluminum layer on the back of the optical addressing potential sensor 152 is connected to the working electrode for outputting a detection signal.
[ Signal Circuit ]
As shown in fig. 4, the signal circuit 120 includes a microprocessor 121 and a LAPS detection circuit 122. The microprocessor 121 implements detection process control using a 16-bit, low power MSP426FG4619 processor. The LAPS detection circuit 122 has a structure including: an I/V conversion circuit 41, a zeroing and amplifying circuit 42, a low-pass filter circuit 43, an impedance circuit 44, and a clock circuit 45. In the LAPS signal detection process, the weak electric signal generated by the optical addressing potential sensor 152 is converted from a photocurrent to a voltage signal by the I/V conversion circuit 41. The I/V conversion circuit 41 with different gear positions can be arranged, and the optimized detection can be realized by matching with an electronic change-over switch. The voltage signal is subjected to signal conditioning through a zeroing and amplifying circuit 42, the direct current bias error of the system is removed, in-phase amplification is performed, the signal to noise ratio is improved, low-frequency noise is filtered through a low-pass filter circuit 43, the signal quality is improved, and the signal is sent to the microprocessor 121. The impedance circuit 44 supplies a high-frequency ac excitation source to the light source modulation circuit 140 for impedance detection, and cooperates to drive the excitation light source, and transmits the detection result to the microprocessor 121 for post-processing. Clock circuit 45 is controlled by microprocessor 121 to provide a stable and reliable clock signal to impedance circuit 44 using ADF4001 clock signal chip and 16M active crystal oscillator.
Example 2
As shown in fig. 5, the microfluidic carbohydrate metabolism analysis detection device 200 of the present embodiment includes: a controller 210, a signal circuit 220, an excitation light source 230, a light source modulation circuit 240, a microfluidic sensor unit 250, a micro syringe pump 260, a waste liquid tank 270, a bubble elimination unit 280, and a vacuum pump 290. The liquid inlet channel of the microfluidic sensor unit 250 is communicated with the microinjection pump 260, the liquid outlet channel of the microfluidic sensor unit 250 is communicated with the waste liquid pool 270, and the microfluidic sensor unit 250 is connected with the signal circuit 220, so that bias voltage and signal acquisition are applied to the microfluidic sensor unit 250. The excitation light source 230 is disposed above the microfluidic sensor unit 250 and connected to the light source modulation circuit 240, so that the microfluidic sensor unit 250 is excited by light to generate photo-generated current. The light source modulation circuit 240 is connected to the signal circuit 220 and is controlled by the signal circuit 220. The signal circuit 220 is connected to the controller 210 through a serial port, and performs the functions of related control, data transmission and processing. A bubble elimination unit 280 is disposed on the liquid inlet channel between the microfluidic sensor unit 250 and the microinjection pump 260, and the bubble elimination unit 280 is communicated with the vacuum pump, and is used for eliminating micro bubbles in the detection solution, thereby improving the detection precision.
The micro syringe pump 260 uses the universal asynchronous receiver/transmitter protocol (UART) to control the syringe pump. The excitation light source 230 is composed of a fixed-focus tunable infrared laser diode module for generating electron hole pairs. The microfluidic carbohydrate metabolism analysis detection device 200 of the present embodiment is identical to the embodiment 1 except for the bubble eliminating unit 280 and the vacuum pump 290, and thus will not be described in detail.
[ bubble eliminating Unit ]
As shown in fig. 6, the bubble eliminating unit 280 of the present embodiment includes: the vacuum pump comprises an upper substrate 61, a lower substrate 62, a negative pressure cavity 63, a fluid cavity 64, a vacuum pump connecting port 65, a micro inflow port 66, a micro outflow port 67, a vacuum pump connecting pipe 68 and a fluid conduit 69, wherein the negative pressure cavity 63 is communicated with the fluid cavity 64 and is positioned between the upper substrate 61 and the lower substrate 62, the negative pressure cavity 63 is adjacent to the upper substrate 61, the fluid cavity 64 is adjacent to the lower substrate 62, the vacuum pump connecting port 65 is positioned on the upper substrate 61 and is communicated with the negative pressure cavity 63, the micro inflow port 66 and the micro outflow port 67 are positioned on the lower substrate 62 and are communicated with the fluid cavity 64. The specific preparation process of the bubble eliminating unit 280 of this embodiment is as follows:
step S610: selecting a p-type silicon wafer with the thickness of 540 mu m, cutting to obtain 2 rectangular silicon single sheets with the thickness of 15mm multiplied by 40mm, cleaning by RCA standard cleaning process, and drying;
step S620: etching the silicon wafer by using negative photoresist, etching a circular groove with the radius of 5mm and the depth of 100 mu m on the silicon single sheet, and forming a plurality of rows of through holes with different diameters, such as through holes with the diameters of 1000 mu m, 500 mu m and 250 mu m, on two sides of the circular groove along the long side direction of the silicon single sheet, wherein the distance between each row of through holes is equal to the diameter of each row of through holes; through holes on the two silicon singlechips correspond to each other (the positions, the diameters and the distances of the through holes are the same), the lengths of the guide grooves are different, the silicon singlechips with short guide grooves are used for forming a negative pressure cavity, and the silicon singlechips with long guide grooves are used for forming a fluid cavity;
step S630: injecting PDMS in a culture dish, uniformly mixing PDMS and a curing agent according to the proportion of 10:1, standing in a vacuum chamber for 20 minutes to remove bubbles, casting into the culture dish, and drying at 80 ℃ for 2 hours to form a PDMS film of 200 mu m;
step S640: a PMMA plate with the thickness of 1mm is selected, 2 rectangular plates with the thickness of 20mm multiplied by 50mm are obtained by cutting, connecting through holes with the diameter of 500 mu m are respectively arranged at positions corresponding to the guiding grooves of the 2 silicon singlechips, fixing through holes matched with the fixing screws are arranged at corners, and the plates are used as an upper substrate 61 and a lower substrate 62 after cleaning and drying;
step S650: sequentially stacking an upper substrate 61, a first silicon single sheet, a PDMS film, a second silicon single sheet and a lower substrate 62, aligning through holes of the first silicon single sheet and through holes of the second silicon single sheet, fixing through holes of the upper substrate 61 and the lower substrate 62, ensuring that connecting through holes of the upper substrate 61 are aligned with guide grooves of the first silicon single sheet, connecting through holes of the lower substrate 62 are aligned with guide grooves of the second silicon single sheet, fixing a screw rod through the fixing through holes of the upper substrate 61 and the lower substrate 62 by nuts, so that a negative pressure cavity 63 is formed between the upper substrate 61 and a circular groove of the first silicon single sheet, a fluid cavity 64 is formed between the PDMS film and the circular groove of the second silicon single sheet, the through holes of the upper substrate 61 are communicated with the negative pressure cavity 63 as vacuum pump connecting ports 65 through the guide grooves of the first silicon single sheet, and the through holes of the lower substrate 62 are respectively communicated with the fluid cavity 64 as micro-inflow ports 66 and micro-outflow ports 67 through the guide grooves of the second silicon single sheet;
step S660: polyethylene pipes having a diameter of 500 μm were respectively used as a vacuum pump connection pipe 68 and a fluid conduit 69, and were fitted into the vacuum pump connection port 65, the micro-inflow port 66 and the micro-outflow port 67, and a sealant was applied to the vacuum pump connection port 65, the micro-inflow port 66 and the micro-outflow port 67.
Example 3
The catabolism of sugars by lactic acid bacteria mainly comprises two pathways, namely the glycolytic pathway (homolactic fermentation) and the phosphoketolase pathway (heterolactic fermentation). The glycolytic pathway (EMP) refers to the glycolysis of glucose under anaerobic conditions to lactate and Adenosine Triphosphate (ATP). The phosphoketolase pathway (phosphoketolase pathway, PK) refers to the glycolysis of glucose under anaerobic conditions to produce equal amounts of lactic acid, ethanol, and CO 2 And ATP. Therefore, lactic acid bacteria can produce lactic acid which affects the taste on sugar metabolism, and the sugar lactic acid bacteria metabolism analysis detection device provided by the application monitors the catabolism condition of the lactic acid bacteria on different sugars by detecting the change of pH. As shown in fig. 7, the present embodiment provides a method for analyzing and detecting carbohydrate metabolism by using the microfluidic carbohydrate metabolism analysis and detection device of embodiments 1 and 2, which comprises the following specific steps:
step S710: calibrating the microfluidic sensor unit and determining a bias voltage range;
a Phosphate Buffered Saline (PBS) with a pH value between 2.0 and 8.0 is used, and a bias voltage of-2.0V to +0.6V is applied, wherein the step voltage is 10mV. The wavelength of excitation light is 450nm, the frequency is 10khz, the I-V curve output by the microfluidic sensor unit is detected in different gradient pH solutions, the photoelectric value at the maximum slope is selected as the working point, and the bias voltage range of the microfluidic sensor unit is determined to be-1.5V to-1.0V.
Step S720: culturing lactobacillus and inoculating into a microfluidic sensor unit;
lactic acid bacteria species in the oral cavity are numerous and mainly found in saliva, dental plaque and oral mucosa, with probiotic properties mainly being lactobacillus, which accounts for about 1% of the total number of oral microorganisms. The experiment was performed using commercially available active lactic acid bacteria of the same species as in the oral cavity. The culture was performed using Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100U/ml penicillin, 100U/ml streptomycin at 37℃and the counts were adjusted to the desired density and inoculated into the microporous membrane 153 of the microfluidic sensor unit. The density of lactobacillus is 0.5-3×10 7 CFU/ml。
Step S730: delivering a sugar solution to the microfluidic sensor unit, applying a bias voltage and modulating excitation light;
the sugar may be a natural sweetener, an artificial sweetener, a single sweetener, a compound sweetener, or a functional sweetener containing other flavoring agents. Artificial sweeteners include high intensity sweeteners and sweet buffers. Other flavoring agents may be sour additives including organic or inorganic acids.
High intensity sweeteners are substances that have a sweetening potency higher than sucrose, fructose or glucose and a lower caloric value. Non-limiting examples include: sodium cyclamate (cyclamate), calcium cyclamate, L- α -aspartyl-N- (2, 4-tetramethyl-3-thiotrimethylene) -D-alaninamide (alitame), aspartame-acesulfame acid, sucralose (sucralose), acesulfame potassium (acesulfame), aspartame (aspartame), saccharin, neohesperidin dihydrochalcone, N- [ N- (3, 3-dimethylbutyl) ] -L- α -aspartic acid-1-methyl ester (neotame), rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, saccharin dulcoside a, stevioside, mogroside iv (mogroside), mogroside v (mogroside), luo han guo sweetener (mogroside), siamenoside, tripotassium glycyrrhizinate, trisodium glycyrrhizinate, monoammonium glycyrrhizinate, ammonium glycyrrhizinate, monopotassium glycyrrhizinate, tripotassium glycyrrhizinate, thaumatin (thaumatin), curculin, monellin, bollin (brazzein), henanthropoxin, gan Chasu, ostutin, verdol, pezoding, cyclamate, glycyrrhizin, acesulfame potassium, aspartame, and the like.
Sweet buffers include substances having a sweetening potency lower than or comparable to sucrose, fructose or glucose, and a lower caloric content, such as sugar alcohols, polyols or polyols in reduced form of sugars, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Non-limiting examples of sweet buffers of the present application include: xylitol, sorbitol, D-mannitol, maltitol, isomalt, erythritol, galactitol, lactitol (4-beta-D-galactopyranose-D-sorbitol); raffinose, lactose, maltose, isomaltulose, alpha-D-glucose, alpha-D-mannose, alpha-D-xylose, alpha-D-galactose, beta-D-fructofuranose, beta-D-maltose, beta-D-lactose; gelatin, sodium caseinate, acacia, tamarind gum, sesbania gum, agar, sodium alginate, potassium alginate, carrageenan, pectin, xanthan gum, beta-cyclodextrin, sodium carboxymethyl cellulose, sodium starch phosphate, sodium carboxymethyl starch, hydroxypropyl starch or propylene glycol alginate.
Organic acid additives include any compound containing a-COOH group including, but not limited to, C2-C30 carboxylic acids, substituted hydroxy C1-C30 carboxylic acids, substituted cinnamic acids, hydroxy acids, substituted hydroxy benzoic acids, substituted cyclohexyl carboxylic acids, tannic acid, lactic acid, tartaric acid, citric acid, fumaric acid, gluconic acid, hydroxycitric acid, malic acid, fumaric acid, maleic acid, succinic acid, salicylic acid, creatine, acetic acid, ascorbic acid, adipic acid, acetic acid, oxalic acid, n-butyric acid, formic acid, polyglutamic acid, glucosamine hydrochloride, glucono-delta-lactone, and alkali metal or alkaline earth metal salt derivatives thereof.
Inorganic acid additives include, but are not limited to, phosphoric acid, phosphorous acid, polyphosphoric acid, carbonic acid, sodium dihydrogen phosphate, and their corresponding alkali or alkaline earth metal salts (e.g., inositol magnesium/calcium hexaphosphate).
The conveying speed of the sugar solution is 300-600mL/h.
Step S740: recording the detection signal in real time, and drawing the lactic acid bacteria metabolism curve of the sugar solution according to the detection signal.
In constant pressure mode, the working temperature is 35-40 ℃, photocurrent signal is continuously recorded for 3-10 minutes, the sensitivity of silicon nitride as a pH value transducer material is 335.5nA/pH, and a time-pH curve is drawn according to the characteristic.
Three passes of the test chamber with deionized water were required between the two sugar solution assays.
[ examples of sugar lactic acid bacteria metabolism detection ]
The bias voltage range is-1.3V, the collection time is 5 minutes, the ultrapure water with the resistivity of more than 15MΩ & cm is used for preparing the solution for testing, and the lactobacillus consumption is 1.0X10 7 CFU/ml, temperature 37 ℃. And measuring and recording volt-ampere characteristic data of each saccharide solution, analyzing to obtain saccharide lactobacillus metabolism characteristic curves, wherein time is taken as an abscissa, and pH change rate is taken as an ordinate. The saccharide solutions tested included:
test example 1:75mmol/L sucrose solution.
Test example 2: a mixed solution with the weight ratio of sucralose, erythritol and maltitol of 3:800:300 and the sweetness consistent with that of a 75mmol/L sucrose solution;
the final test curve results show that the pH of the fermentation solution of sucrose solution and lactobacillus gradually decreases with the passage of time, and the rate of decrease of the pH value increases with the increase of the amount of lactic acid generated by the reaction. After 40-60 seconds, the pH value starts to rapidly decrease, the pH decrease gradually slows down in 120-180 seconds, and the pH value is stabilized between 3 and 4. In contrast, the artificial sweetener solution had less fermentation reaction with lactic acid bacteria and only a small drop in pH.
[ simulation example of taste of sugar metabolism ]
The bias voltage range is-1.3V, the collection time is 5 minutes, the ultrapure water with the resistivity of more than 15MΩ & cm is used for preparing the solution for testing, and the lactic acid bacteria is usedThe amount was 1.0X10 7 CFU/ml, temperature 36 ℃. And measuring and recording volt-ampere characteristic data of each saccharide solution, analyzing to obtain saccharide lactobacillus metabolism characteristic curves, wherein time is taken as an abscissa, and pH change rate is taken as an ordinate. The saccharide solutions tested included:
standard solution: 75mmol/L sucrose solution.
The specific contents of the components of the acidity gradient test solution are shown in table 1.
Table 1: content of test liquid component
The sample pH was measured using a Digital waterproof pH meter, HM Digital pH meter pH-200. The pH meter was calibrated with a standard pH7.0 reference solution produced by General Hydroponics. The device is calibrated prior to measurement.
The results show that the test liquid with the sugarcane acidity ranging from 3.0 to 4.0 has higher matching degree with the sucrose metabolism in 120 to 180 seconds with the time.
While the intent and embodiments of the present application have been described in detail by way of examples, those skilled in the art to which the application pertains will appreciate that the foregoing examples are merely illustrative of the preferred embodiments of the present application, and that it is not intended to list all embodiments individually and that any implementation embodying the technical scheme of the present application is within the scope of the present application.
It should be noted that the above description of the present application is further detailed in connection with the specific embodiments, and it should not be construed that the specific embodiments of the present application are limited thereto, and those skilled in the art can make various improvements and modifications on the basis of the above-described embodiments while falling within the scope of the present application.
Claims (15)
1. A microfluidic carbohydrate metabolic analysis detection device, comprising: the micro-fluidic device comprises a micro-fluidic sensor unit, a micro-injection pump, a waste liquid pool, a signal circuit, an excitation light source, a light source modulation circuit and a controller;
the liquid inlet channel of the microfluidic sensor unit is communicated with the microinjection pump, and the liquid outlet channel of the microfluidic sensor unit is communicated with the waste liquid pool; the microfluidic sensor unit comprises a substrate, an optical addressing potential sensor, a microporous membrane, a liquid inlet pipe, a liquid outlet pipe and a working electrode; the conducting layer of the optical addressing potential sensor is connected with the working electrode, and the optical addressing potential sensor and the working electrode are arranged on the substrate; the microporous membrane is arranged on the optical addressing potential sensor; has a micro-flow groove; the liquid inlet pipe and the liquid outlet pipe are arranged at two ends of the micro-groove and are inserted into the micro-porous membrane;
the signal circuit is connected with the microfluidic sensor unit, applies bias voltage to the microfluidic sensor unit and collects detection signals from the microfluidic sensor unit, and is connected with the light source modulation circuit to send modulation control signals;
the excitation light source is arranged towards the microfluidic sensor unit and connected with the light source modulation circuit, and emits excitation light for generating photocurrent to the microfluidic sensor unit;
the light source modulation circuit is connected with the excitation light source and modulates the excitation light source to emit excitation light according to the modulation control signal;
the controller is in communication connection with the signal circuit and is used for detecting, controlling, recording and processing the acquired data.
2. The microfluidic carbohydrate metabolism analysis detection device according to claim 1, wherein: the micro-launder is in a spinning vertical shape.
3. The microfluidic carbohydrate metabolism analysis detection device according to claim 1, wherein: the liquid inlet pipe and the liquid outlet pipe are composed of a metal pipe and a polyethylene microporous pipe, the metal pipe is inserted into the microporous membrane, and the polyethylene microporous pipe is sleeved on the metal pipe.
4. A microfluidic carbohydrate metabolism analysis detection device according to claim 3, wherein: the metal tube is made of stainless steel, silver or platinum.
5. A microfluidic carbohydrate metabolism analysis detection device according to claim 3, wherein: the metal tube of the liquid inlet tube or the metal tube of the liquid outlet tube is used as a reference electrode.
6. The microfluidic carbohydrate metabolism analysis detection device according to claim 1, wherein: the optical addressing potential sensor is sequentially composed of an aluminum layer, a silicon wafer substrate, an insulator layer and a protective layer from the back surface to the front surface.
7. The microfluidic carbohydrate metabolism analysis detection device according to claim 6, wherein:
the aluminum layer is connected with the working electrode, the protective layer is positioned below the microporous membrane, and the protective layer exposed by the micro-groove is arranged towards the excitation light source.
8. The microfluidic carbohydrate metabolism analysis detection device according to claim 1, wherein: the signal circuit comprises a microcontroller and a LAPS detection circuit;
the LAPS detection circuit comprises an I/V conversion circuit, a zeroing and amplifying circuit, a low-pass filter circuit, an impedance detection circuit and a clock circuit; the photocurrent signal of the optical addressing potential sensor is converted into a voltage signal through the I/V conversion circuit; the voltage signal is subjected to error removal and in-phase amplification through the zeroing and amplifying circuit, low-frequency noise is filtered through the low-pass filtering circuit, and the low-frequency noise is sent to the microcontroller; the impedance detection circuit provides a high-frequency alternating current excitation source for the light source modulation circuit to perform impedance detection and drive an excitation light source; the clock circuit provides a clock signal for the impedance detection circuit through microprocessor control.
9. The microfluidic carbohydrate metabolism analysis detection device according to claim 1, wherein: and a bubble eliminating unit is arranged on the liquid inlet channel and is communicated with a vacuum pump.
10. The microfluidic carbohydrate metabolism analysis detection device according to claim 9, wherein: the bubble eliminating unit comprises an upper substrate, a lower substrate, a negative pressure cavity, a fluid cavity, a vacuum pump connecting port, a micro-inflow port, a micro-outflow port, a vacuum pump connecting pipe and a fluid conduit;
the negative pressure cavity is communicated with the fluid cavity, the negative pressure cavity and the fluid cavity are positioned between the upper substrate and the lower substrate, the negative pressure cavity is adjacent to the upper substrate, and the fluid cavity is adjacent to the lower substrate;
the vacuum pump connecting port is positioned on the upper substrate and communicated with the negative pressure cavity;
the micro-inflow port and the micro-outflow port are positioned on the lower substrate and are communicated with the fluid cavity.
11. A method for performing a carbohydrate metabolism analysis assay using the device of any of claims 1-10, the method comprising:
calibrating the microfluidic sensor unit and determining a bias voltage range;
culturing lactobacillus and inoculating into a microfluidic sensor unit;
delivering a sugar solution to the microfluidic sensor unit, applying a bias voltage and modulating excitation light;
recording the detection signal in real time, and drawing the lactic acid bacteria metabolism curve of the sugar solution according to the detection signal.
12. The method for analytical detection of carbohydrate metabolism according to claim 11, wherein: the bias voltage ranges from-1.5V to-1.0V; the lactobacillus has density of 0.5-3×10 7 CFU/ml。
13. The method for analytical detection of carbohydrate metabolism according to claim 11, wherein: the conveying speed of the sugar solution is 300-600mL/h; the detection temperature is 35-40 ℃.
14. The method for analytical detection of carbohydrate metabolism according to claim 11, wherein: the sugar solution comprises 75mmol/L sucrose solution and mixed solution containing sucralose, erythritol and maltitol, and having sweetness consistent with that of the 75mmol/L sucrose solution.
15. The method for analytical detection of carbohydrate metabolism according to claim 11, wherein: the sugar solution comprises 75mmol/L sucrose solution and mixed solution containing sucralose, erythritol, maltitol and vitamin C, wherein the sweetness of the mixed solution is consistent with that of the 75mmol/L sucrose solution, and the acidity of the mixed solution is between pH3.0 and 4.0.
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