AU2021100539A4

AU2021100539A4 - Terahertz biosensor, and preparation method and use thereof

Info

Publication number: AU2021100539A4
Application number: AU2021100539A
Authority: AU
Inventors: Lanlan FAN; Tiejun Li; Jianjun Liu; Guifang SHAO
Original assignee: Shaoguan University
Current assignee: Shaoguan University
Priority date: 2020-05-11
Filing date: 2021-01-28
Publication date: 2021-04-15
Anticipated expiration: 2029-01-28
Also published as: CN111781158B; CN111781158A

Abstract

The present disclosure provides a terahertz biosensor, and a preparation method and use thereof, and belongs to the technical field of terahertz biosensors. The terahertz biosensor includes a terahertz differential microfluidic plate channel. The terahertz differential microfluidic plate channel includes an inlet channel, a first microfluidic plate, a second microfluidic plate and an outlet channel that are successively connected; the first microfluidic plate and the second microfluidic plate are connected via a microchannel; the terahertz differential microfluidic plate channel adopts quartz glass as a substrate and polydimethylsiloxane (PDMS) as a cover film; and the surface of the quartz glass is grafted with P(NIPAAm-co-VPBA) having thermoresponsive boronate affinity. The terahertz biosensor combines terahertz characteristics with microfluidic technology to achieve highly-sensitive isolation and detection of cis-dihydroxy biomolecules. 2 /7 N C 2 FIG. 2

Description

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FIG. 2

TERAHERTZ BIOSENSOR, AND PREPARATION METHOD AND USE THEREOF

TECHNICAL FIELD The present disclosure belongs to the technical field of terahertz biosensors, and in particular to a terahertz biosensor, and a preparation method and use thereof.

BACKGROUND With the development of genetic engineering technology, human beings can insert foreign genes into an organism, so that the organism can have new characteristics such as insect resistance and stress resistance while retaining its own characteristics. However, the transgenic species may pose potential threats to human health, ecological environment, ethics, and so on. Therefore, it is necessary to detect and identify genetically modified organisms (GMOs). Genetically-modified substances are characterized by multiple types, complex composition and low transgene content. Therefore, highly-sensitive and rapid detection of target proteins in GMO samples is the basis for subsequent research. At present, common detection methods, which are time-consuming, labor-intensive, complicated and costly, will destroy proteins or gene fragments to a certain extent and can hardly be conducted by non-professionals. Therefore, the methods are not suitable for highly-sensitive and real-time on-line detection of genes and proteins. Studies have found that the characteristic vibration modes of many biological macromolecules (such as proteins or DNA) are located in terahertz frequency band, making terahertz a potential biochemical sensing tool. However, water exhibits high absorptive capacity to terahertz radiation, which makes it relatively difficult to study the terahertz transmission spectrum of a liquid sample. Therefore, it is necessary to find a method that can shorten the interaction length of a liquid sample with terahertz to reduce the strong absorption of water to terahertz radiation. Microfluidic technology has been listed as one of the most important frontier technologies in the 21st century. Microfluidic technology, as a potential analysis platform, can be integrated with the biotechnology or analysis method used in the laboratory. Microfluidic technology can be used to precisely control the thickness of a liquid within 100 [m, which can reduce the consumption of a sample, shorten the analysis time, improve the sensitivity of detection, and realize the high-throughput detection of a sample. Therefore, the combination of terahertz and microfluidic technology will provide an interesting direction for studying the characteristics of biosensors. However, water in a sample exhibits high absorptive capacity to terahertz radiation during detection, and detection error is easily caused by the non-sensing drift interference in the terahertz spectral detection. Therefore, it is unable to achieve the highly-sensitive detection of protein. SUMMARY In view of this, the present disclosure is intended to provide a terahertz biosensor, and a preparation method and use thereof. In the present disclosure, the terahertz biosensor combines terahertz characteristics with microfluidic technology, where, a differential microfluidic plate channel structure is designed for sensing on a microfluidic chip with quartz glass as a substrate to avoid error caused by non-sensing drift interference in the terahertz spectral detection. Further, a bovine serum albumin (BSA) coating is introduced into a microfluidic channel to avoid the adsorption of the inner wall of the microfluidic channel to a target protein in an analyte, so as to minimize the non-specific adsorption of the inner wall of the microfluidic channel to a biological sample, thereby realizing regeneration of the modification of the inner wall of the microfluidic channel. Further, surface-initiated atom transfer radical polymerization (SI-ATRP) is used to graft P(NIPAAm-co-VPBA) with thermoresponsive boronate affinity in the glass microfluidic channel, the hydrophilic and hydrophobic states of the P chain are controlled by temperature regulation to realize the enrichment and release of a target protein, and terahertz time-domain spectroscopy (THz-TDS) is adopted for characterization, thereby realizing real-time, high-throughput and highly-sensitive detection of protein. The present disclosure provides a terahertz biosensor, including a terahertz differential microfluidic plate channel; The terahertz differential microfluidic plate channel includes an inlet channel, a first microfluidic plate, a second microfluidic plate and an outlet channel that are successively connected; the first microfluidic plate and the second microfluidic plate are connected via a microchannel; the terahertz differential microfluidic plate channel adopts quartz glass as a substrate and polydimethylsiloxane (PDMS) as a cover film; and the surface of the quartz glass is grafted with P(NIPAAm-co-VPBA) having thermoresponsive boronate affinity. Preferably, the terahertz differential microfluidic plate channel may have a depth of 40 pm to 60 m. Preferably, the first and second microfluidic plates may have a spacing of 20 m to 40

[m. Preferably, the microchannel may have a width of 10 m to 100 [m. Preferably, the inner wall of the terahertz differential microfluidic plate channel may be provided with a BSA coating. Preferably, a coating agent used for the BSA coating may have a BSA concentration of 1 mg/mL to 2 mg/mL; and the coating agent may have a pH of 4.0 to 5.0. The present disclosure further provides a method for preparing the terahertz biosensor, including the following steps: 1) grafting P(NIPAAm-co-VPBA) on the surface of a quartz glass substrate by SI-ATRP reaction to obtain a glass substrate with thermoresponsive boronate affinity; 2) subjecting an embossed mask with a differential microfluidic plate channel structure to photoetching and photocuring using SU-8 photoresist under the action of ultraviolet (UV), and rinsing to obtain a mask with a photoresist layer; 3) bonding the mask with a photoresist layer and the glass substrate with thermoresponsive boronate affinity, and removing the mask to obtain a substrate of the terahertz differential microfluidic plate channel; and 4) bonding the substrate of the terahertz differential microfluidic plate channel with an SU-8 photoresist cover sheet to obtain the terahertz differential microfluidic plate channel, where, the SU-8 photoresist cover sheet may have an inlet channel and an outlet channel. Preferably, the method may further include: injecting the coating agent into the terahertz differential microfluidic plate channel for coating, and standing a resulting coating. Preferably, the standing may be conducted for 5 h to 10 h. Preferably, the photoetching and photocuring may be conducted for 30 s to 35 s. The present disclosure further provides use of the terahertz biosensor in the isolation and detection of cis-dihydroxy biomolecules. Preferably, the cis-dihydroxy biomolecules may include proteins and nucleic acids. Preferably, the proteins may include genetically-modified Bacillus thuringiensis (BT) protein. Preferably, a to-be-tested sample may be passed through the terahertz biosensor, and a target may be captured at 4°C to 55°C and released at a temperature < 4°C. Preferably, THz-TDS may be adopted in a frequency range of 0.3 THz to 1.1 THz for characterization, and the type of the target is determined according to characterization results. Preferably, the difference between terahertz spectral responses measured by the first and second microfluidic plates may be calculated to obtain the final terahertz spectral response value. The present disclosure has the following beneficial effects: The terahertz biosensor provided in the present disclosure combines terahertz characteristics with microfluidic technology to achieve highly-sensitive isolation and detection of cis-dihydroxy biomolecules. The present disclosure analyzes the factors causing unstable detection results of cis-dihydroxy biomolecules from two aspects of terahertz and microfluidics, so as to establish a method that can limit the strong absorption of water to terahertz and eliminate non-sensing drift interference. At present, research on detection of cis-dihydroxy biomolecules using terahertz is mostly limited to the algorithm design or improvement to increase the processing accuracy, but pays little attention to the factors affecting detection results. The present disclosure is distinguished from the previous biological detection methods by considering the influence and evaluation on detection results, where, research is conducted from the two aspects of terahertz and microfluidics to combine the respective characteristics, and a differential microfluidic plate channel structure is designed for sensing on a microfluidic chip with quartz glass as a substrate to inhibit the strong absorption of water to terahertz and to avoid error caused by non-sensing drift interference in the terahertz spectral detection, thereby improving the stability of detection results. In the present disclosure, in order to reduce the interaction between a protein and the inner wall of the microfluidic plate channel, a BSA coating is applied to the inner wall of the microfluidic plate channel to inhibit the non-specific adsorption of the inner wall of microfluidic plate channel to biomolecules. In general, biomolecules, such as proteins, are easily adsorbed on the inner surface of the microfluidic plate channel, which can lead to poor peak shape, unrepeatable migration time, and irregular electro-osmotic flow (EOF). Therefore, one of the main tasks in the detection based on the terahertz differential microfluidic plate is to reduce the interaction of a to-be-tested substance with the inner wall of the microfluidic plate channel. In order to avoid the disadvantages of coating directly in the microfluidic plate channel, the present disclosure provides a simple, rapid and effective coating method for introducing a BSA coating into the microfluidic plate channel, and the BSA coating can minimize the non-specific adsorption of the inner wall of microfluidic plate channel to a protein sample. In order to increase the relative concentration of a target protein, the present disclosure provides a target protein capture and release method based on thermoresponsive boronate affinity. How to achieve highly-sensitive and rapid detection of a target protein in various biological samples with a complex composition and a low target content has always been one of the scientific problems which analysts are dedicated to pursue. Therefore, it is of practical significance to achieve the locking of a low-concentration target protein. In the present disclosure, before detection, a sample is pre-treated to increase the concentration of a target substance, and to purify and isolate the target substance from a mixture. In the present disclosure, SI-ATRP is used to graft P(NIPAAm-co-VPBA) in the terahertz microfluidic plate channel, and the hydrophilic and hydrophobic states of the P(NIPAAm-co-VPBA) chain are controlled by temperature regulation, thereby realizing the enrichment and release of a target protein. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a block diagram illustrating the overall design of a transgenesis terahertz biosensor;

FIG. 2 is a schematic diagram (not to scale) for the prototype of a differential microfluidic channel, where, the left is a top view and the right is a sectional view; FIG. 3 shows the absorption coefficient (gray line) and refractive index (black line) of water in the frequency range of 0.3 THz to 1.1 THz; FIG. 4 shows the isolation effect of a glass microfluidic chip with a BSA coating on the inner wall thereof for protein; FIG. 5 shows the principle of grafting P(NIPAAm-co-VPBA) on the surface of a quartz glass substrate by SI-ATRP reaction; FIG. 6 is a flow chart for preparing a microfluidic plate channel with thermoresponsive boronate affinity; FIG. 7 shows the principle of capturing and releasing a target by the microfluidic plate channel with thermoresponsive boronate affinity; FIG. 8 shows the measurement of contact angle of p graft with the surface of the glass substrate at different temperatures, where, (a) 4°C, (b) 25°C, and (c) 55°C; and FIG. 9 shows THz frequency-domain spectra of three cotton varieties obtained in an embodiment. DETAILED DESCRIPTION The present disclosure provides a terahertz biosensor, including a terahertz differential microfluidic plate channel. The terahertz differential microfluidic plate channel includes an inlet channel, a first microfluidic plate, a second microfluidic plate and an outlet channel that are successively connected; the first microfluidic plate and the second microfluidic plate are connected via a microchannel; the terahertz differential microfluidic plate channel adopts quartz glass as a substrate and polydimethylsiloxane (PDMS) as a cover film; and the surface of the quartz glass is grafted with P(NIPAAm-co-VPBA) having thermoresponsive boronate affinity. The overall design idea Si is shown in Figure 1 and is as follows: guiding S103 terahertz characteristic spectrum extraction indexes through method comparison S101 and theoretical analysis S102 (including reducing the absorption of water to terahertz radiation S104 and eliminating non-sensing interference S105) to form an evaluation system S107, acquiring parameter extraction S108, preparing a channel S109, building a platform S110, and performing genetic testing SI11. A specific design framework S2 includes parameter selection and structural design S201; and highly-sensitive detection of introduced Bt gene using terahertz S205 is achieved through the preparation of a terahertz differential microfluidic plate channel S202, the modification of the inner wall of the microfluidic plate channel S203, and the enrichment and isolation of a target protein S204. In the present disclosure, the structure of the terahertz differential microfluidic plate channel is shown in FIG. 2. In the present disclosure, the terahertz differential microfluidic plate channel may have a depth preferably of 40 m to 60 m, and more preferably of 45 [m to 55 m. The depth of the terahertz differential microfluidic plate channel is set to the above range to control the interaction length of a liquid sample with terahertz. In the present disclosure, the first and second microfluidic plates may have a spacing preferably of 20 m to 40 m, and more preferably of 25 m to 35 m. The spacing between the first and second microfluidic plates is controlled within the above range to effectively eliminate non-sensing drift interference. In the present disclosure, the microchannel may have a width preferably of 10 m to 100 jm, and the width of the microchannel can be adjusted according to the size of a to-be-tested biomolecule and the amount of a liquid sample. In the present disclosure, the inner wall of the terahertz differential microfluidic plate channel may preferably be provided with a BSA coating. In the present disclosure, a coating agent used for the BSA coating may have a BSA concentration preferably of 1 mg/mL to 2 mg/mL, and more preferably of 1.6 mg/mL; the coating agent may have a pH preferably of 4.0 to 5.0, and more preferably of 4.3; and a solvent for the coating agent may preferably be phosphate buffer (PB). The present disclosure adopts RNase A as an isolation object, and the highest theoretical plate number can be achieved for RNase A when the BSA coating agent has a BSA concentration of 1.6 mg/mL and a pH of 4.3, reaching 1.28 x 105. The present disclosure further provides a method for preparing the terahertz biosensor, including the following steps: 1) grafting P(NIPAAm-co-VPBA) on the surface of a quartz glass substrate by SI-ATRP reaction to obtain a glass substrate with thermoresponsive boronate affinity; 2) subjecting an embossed mask with a differential microfluidic plate channel structure to photoetching and photocuring using SU-8 photoresist under the action of UV, and rinsing to obtain a mask with a photoresist layer; 3) bonding the mask with a photoresist layer and the glass substrate with thermoresponsive boronate affinity, and removing the mask to obtain a substrate of the terahertz differential microfluidic plate channel; and 4) bonding the substrate of the terahertz differential microfluidic plate channel with an SU-8 photoresist cover sheet to obtain the terahertz differential microfluidic plate channel, where, the SU-8 photoresist cover sheet may have an inlet channel and an outlet channel.

In the present disclosure, P(NIPAAm-co-VPBA) is grafted on the surface of a quartz glass substrate by SI-ATRP reaction to obtain a glass substrate with thermoresponsive boronate affinity. In the present disclosure, the principle of the SI-ATRP reaction is shown in FIG. 5. In the present disclosure, P polymer is grafted on a quartz glass substrate and a microfluidic plate channel by SI-ATRP under UV induction, and the specific process may include the following steps: (1) soaking a quartz glass substrate in an acetone solution with methoxyphenone for 1 h, and rinsing the quartz glass substrate with deionized water; (2) adding a graft mixed solution dropwise on the quartz glass substrate for grafting under UV irradiation, and after the UV grafting polymerization is completed, rinsing successively with acetone and water to obtain a glass substrate with thermoresponsive boronate affinity. In the present disclosure, methoxyphenone in the acetone solution with methoxyphenone may have a volume fraction preferably of 20%; and the rinsing with deionized water may preferably be conducted as follows: rapidly rinsing with sufficient deionized water. In the present disclosure, the graft mixed solution may preferably include the following components: 10% (w/v) NIPAAm, 8.44 mg PBA (a molar ratio of NIPAAm to PBA: 10:1), 0.5mM NaIO4, and 0.5% (w/v) benzyl alcohol. In the present disclosure, the graft mixed solution may be added dropwise at a volume preferably of 150 L to 250 [L, and more preferably of 200 [L. In the present disclosure, the quartz glass substrate added with the graft mixed solution may preferably be placed on the ice to dissipate the heat generated during the reaction initiated by UV irradiation. In the present disclosure, the UV irradiation grafting may preferably be realized through the UV irradiation on a photoetching machine; and the UV irradiation grafting may be conducted preferably for 30 s to 35 s, and more preferably for 32 s. In the present disclosure, after the UV grafting polymerization is completed, the quartz substrate may be fully rinsed with a large amount of acetone and water successively. In the present disclosure, an embossed mask with a differential microfluidic plate channel structure is subjected to photoetching and photocuring using SU-8 photoresist under the action of UV, and then rinsed to obtain a mask with a photoresist layer. In the present disclosure, the embossed mask with a differential microfluidic plate channel structure may preferably be made from quartz; the embossed mask may be prepared preferably by a positive photoresist process; and the preparation of the embossed mask may preferably be entrusted to a mask production company. In a specific implementation of the present disclosure, a mask frame is fixed on a chrome plate, and the degassed SU-8 photoresist is spread in the mask frame; and the embossed mask with a differential microfluidic plate channel structure is placed on the SU-8 photoresist in the mask frame for photoetching and photocuring. In the present disclosure, it is preferably to avoid the formation of bubbles between the embossed mask and the SU-8 photoresist; and the photoetching and photocuring may be conducted preferably for 30 s to 35 s, and more preferably for 32 s. In the present disclosure, after the photoetching and photocuring is completed, the mask and the cured photoresist may preferably be torn off from the chrome plate and then rinsed. The rinsing may preferably be conducted using a photoresist-cleaning solution. After the rinsing, the mask may preferably be blow dried with a blower. The rinsing and drying process may preferably be conducted twice. In the present disclosure, the mask with a photoresist layer is bonded with the glass substrate having thermoresponsive boronate affinity, and the mask is then removed to obtain a substrate of the terahertz differential microfluidic plate channel. In the present disclosure, the bonding may preferably be conducted at a pressure of 8.64 N; a bond head for the bonding may preferably have a temperature of 280°C; and the bonded glass substrate may preferably have a temperature of 100°C. In the present disclosure, after the bonding, a photoresist gun may preferably be used to seal the edge, and the upper mask may be torn off to obtain a substrate of the terahertz differential microfluidic plate channel. In the present disclosure, the substrate of the terahertz differential microfluidic plate channel is bonded with an SU-8 photoresist cover sheet to obtain the terahertz differential microfluidic plate channel. In the present disclosure, the SU-8 photoresist cover sheet may preferably have an inlet channel and an outlet channel; and the preparation method of the SU-8 photoresist cover sheet is similar to the preparation method of the substrate of the terahertz differential microfluidic plate channel, as shown in the right panel of FIG. 6, which will not be repeated here. In the present disclosure, the bonding may preferably be conducted at a pressure of 8.64 N; a bond head for the bonding may preferably have a temperature of 280°C; and the bonded glass substrate may preferably have a temperature of 100°C. In the present disclosure, after the terahertz differential microfluidic plate channel is obtained, the method may further include: injecting the coating agent into the terahertz differential microfluidic plate channel for coating, and standing a resulting coating. In the present disclosure, the standing may be conducted preferably for 5 h to 10 h. In the present disclosure, the coating agent may preferably be injected by a peristaltic pump. The present disclosure further provides use of the terahertz biosensor in the isolation and detection of cis-dihydroxy biomolecules. In the present disclosure, the cis-dihydroxy biomolecules may preferably include proteins and nucleic acids; and the proteins may preferably include genetically-modified BT protein. In the present disclosure, the use may preferably include the following steps: a to-be-tested sample is passed through the terahertz biosensor, and a target may be captured at

4°C to 55°C and released at a temperature < 4°C. The present disclosure realizes the capture and release of a target by temperature regulation, so as to enrich and isolate the target in a to-be-tested sample. The present disclosure can achieve the detection of the target after the target is released. In the present disclosure, THz-TDS may preferably be adopted in a frequency range of 0.3 THz to 1.1 THz for characterization, and the type of the target is determined according to characterization results. In the present disclosure, the difference between terahertz spectral responses measured by the first and second microfluidic plates may preferably be calculated to obtain the final terahertz spectral response value. The technical solutions provided by the present disclosure will be described in detail below with reference to embodiments, but the embodiments should not be construed as limiting the claimed scope of the present disclosure. Embodiment 1 A terahertz biosensor includes a terahertz differential microfluidic plate channel. The terahertz differential microfluidic plate channel includes an inlet channel, a first microfluidic plate, a second microfluidic plate and an outlet channel that are successively connected; the first microfluidic plate and the second microfluidic plate are connected via a microchannel; the terahertz differential microfluidic plate channel adopts quartz glass as a substrate and PDMS as a cover film; and the surface of the quartz glass is grafted with P(NIPAAm-co-VPBA) having thermoresponsive boronate affinity. The structure of the terahertz differential microfluidic plate channel is shown in FIG. 2. In the present disclosure, the terahertz differential microfluidic plate channel may have a depth preferably of 50 m; the first and second microfluidic plates may have a spacing preferably of 30 m; and the microchannel may have a width preferably of 50 m, and the width of the microchannel can be adjusted according to the size of a to-be-tested biomolecule and the amount of a liquid sample. The inner wall of the terahertz differential microfluidic plate channel may be provided with a BSA coating. A coating agent used for the BSA coating may have a BSA concentration of 1.6 mg/mL; the coating agent may have a pH of 4.3; and a solvent for the coating agent may be PB. The present disclosure adopts RNase A as an isolation object, and the highest theoretical plate number can be achieved for RNase A when the BSA coating agent has a BSA concentration of 1.6 mg/mL and a pH of 4.3, reaching 1.28 X 105.

Preparation Method: The present disclosure adopted the finite element analysis method to set the parameters and boundary conditions of the microfluidic plate channel and initially established a differential microfluidic plate channel under the COMSOL Multiphysics platform. In the present disclosure, quartz glass with low absorption rate was adopted as a substrate, and PDMS easy to adhere and manufacture was adopted as a cover film. In the present disclosure, before detection, a sample was pre-treated by the enrichment and isolation technology, to purify and isolate the target substance from a mixture. With the change of temperature, the polymer chain of the thermoresponsive polymer P(NIPAAM-co-PBA) formed by poly(N-isopropylacrylamide) (PNIPAAm) and phenylboronic acid (PBA) exhibited transition between hydrophilic and hydrophobic properties, characterized by stretching and coiling of the polymer chain, so as to achieve the capture and release of cis-dihydroxy-containing biomolecules. P polymer was grafted on the quartz glass substrate and the microfluidic plate channel by SI-ATRP under UV induction, and the principle was shown in FIG. 5. The specific process of grafting on the quartz glass substrate was as follows (similar to the grafting in the microfluidic plate channel): (1) a quartz glass substrate was soaked in an acetone solution with 20% of methoxyphenone for a specified period of time, and rapidly rinsed with sufficient deionized water; (2) 1 mL of a mixed solution including 10% (w/v) NIPAAm, 8.44 mg PBA (a molar ratio of NIPAAm to PBA: 10:1), 0.5 mM NaIO4 and 0.5% benzyl alcohol (w/v) was prepared; (3) 200 L of the mixed solution was pipetted and added dropwise to the center of the surface of the quartz glass substrate, and then the quartz substrate was placed on a plastic box filled with ice to dissipate the heat generated during the reaction initiated by UV radiation; (4) the quartz substrate was irradiated with UV light on a photoetching machine; and (5) after the UV grafting polymerization was completed, the quartz substrate was fully rinsed with a large amount of acetone and water successively. The specific preparation process of a microfluidic plate channel with thermoresponsive boronate affinity was shown in FIG. 6. The embossed mask and cover sheet mask of the microfluidic channel (only with an inlet and outlet) and the glass substrate grafted with P polymer were prepared on PDMS, and then used to construct a microfluidic microchip. The microfluidic chip was constructed as follows: (1) a mask frame was fixed on a chrome plate; (2) 400 L of degassed SU-8 photoresist was drawn by a syringe and then gently spread in the mask frame; (3) an embossed mask with a differential microfluidic plate channel structure was gently placed on the SU-8 photoresist in the mask frame, ensuring no bubble is formed between the mask and the SU-8 photoresist; (4) the chrome plate was placed under a photoetching machine for 32 s of photoetching and photocuring, where, only the position with the mask channel was not cured; (5) the mask and the cured photoresist layer were gently torn off from the chrome plate, and the cured photoresist layer on the mask was quickly rinsed with a photoresist-cleaning solution and then dried with a blower, the rinsing and drying process was repeated twice; (6) the photoresist layer was rapidly bonded with the glass substrate with thermoresponsive boronate affinity prepared above, and then a photoresist gun was used to seal the edge; and after the bonding was firm, the upper mask was torn off; (7) the same method was used to prepare a cover sheet of the photoresist layer channel, where, the cover sheet was an SU-8 substrate with only an inlet and an outlet; and (8) the cover sheet was bonded with the SU-8 substrate bonded on the glass substrate to prepare a microfluidic chip with a differential microfluidic plate and thermoresponsive boronate affinity. Inner wall coating of the terahertz differential microfluidic plate channel and stability evaluation thereof (1) Inner wall coating of the terahertz differential microfluidic plate channel In the detection of genetically-modified substances, proteins, especially genetically-modified proteins, are easily adsorbed to the inner wall surface of the microfluidic plate channel. This adsorption is mainly caused by the electrostatic interaction between proteins and the inner wall of the microfluidic plate channel. Studies have found that non-specific adsorption for proteins may lead to poor peak shape, unrepeatable migration time, and irregular EOF, thereby resulting in unreliable gene expression profiling (GEP) data and uninterpretable or meaningless analysis results for gene expression data. In view of this, the present disclosure modifies the inner wall of the microfluidic plate channel by introducing a BSA coating and characterizes the coating. A chemically-common mixed solution with ribonuclease (RNase A) and dimethyl sulfoxide (DMSO) may be adopted as an analyte for optimizing and evaluating the coating, and the peak shape and theoretical plate number of RNase A chromatographic peak and the migration time of DMSO may be adopted to evaluate the performance of the coated capillary. Formula of theoretical plate number: where, N represents the migration time of RNase A (min); and W1/2 represents the half-width (cm). Because the preparation of a quartz glass microfluidic chip is cumbersome and takes a long time, and the quartz glass microfluidic chip is more expensive than capillaries of the same material, directly conducting coating and characterization repeatedly in the microfluidic chip will cause waste of microfluidic chips, which is also time-consuming and labor-consuming. In addition, when the coated microfluidic chip is characterized, electrophoresis is usually conducted for fluorescein isothiocyanate (FITC)-labeled proteins, which requires fluorescent labeling of proteins, resulting in higher consumption of time and reagents. Since the capillary and the glass chip are made of the same material, coating the inner wall of the capillary and coating the channel of the chip are the same in principle, and the sealing effects after coating are also the same. Moreover, the capillary electrophoresis (CE) instrument used in CE is a UV detector, which can directly conduct automated electrophoresis to characterize the coating effect. In order to avoid the above drawbacks of directly coating in the microfluidic chip, the present disclosure first conducted BSA coating on the inner wall of the capillary by flushing with a peristaltic pump, then characterized the coating after standing for a specified period of time, and finally found the optimal coating conditions and applied the conditions to the microfluidic chip. As shown in FIG. 4, the glass microfluidic chip achieved the isolation of the standard protein mixture of catalase and carbonic anhydrase after being modified with the BSA coating on the inner wall thereof. In the optimization process, it was necessary to examine the pH of the coating buffer, the standing time of the coating, and the BSA concentration of the coating to determine the optimal coating buffer pH and coating BSA concentration, which were applied to the microfluidic plate channel to achieve the purpose of reducing the interaction between a protein and the inner wall of the microfluidic chip and avoiding the adsorption of the inner wall of the microfluidic plate channel to a target protein in an analyte. Since the isoelectric point (pI) of BSA is 4.7, a buffer with a pH of 4.0 to 7.0 was adopted as the coating buffer. Positively and negatively charged BSA coating buffers were prepared, separately. The BSA coating buffer was PB. At the same time, a series of BSA coating solutions with different concentrations of 0.2 mg/mL, 0.70 mg/mL, 1.6 mg/mL, 2 mg/mL, 5 mg/mL, 10 mg/mL, and 15 mg/mL were prepared. CE experimental verification was conducted for BSA-coated capillaries. Results obtained from the experimental verification were as follows: a) It can be seen from the peak shape verification of chromatographic peak that, when the coating buffer pH was 4.3, the RNase A peak and the DMSO peak had been completely separated, and the peak shape of RNaseA was the best at all different coating BSA concentrations. Therefore, 4.3 was adopted as the optimal buffer pH for the coating. b) It can be seen through the calculation of theoretical plate number that, when the coating had a BSA concentration of 1.6 mg/mL and the optimal coating buffer pH was 4.3, the theoretical plate number for RNase A was the highest, reaching 1.28 x 105. Therefore, 1.6 mg/mL was adopted as the optimal concentration for BSA coating. c) Under the optimal coating buffer pH of 4.3 and the optimal coating BSA concentration of 1.6 mg/mL, the standing time for the coating was optimized. BSA coating was conducted for four identical capillaries under the same conditions except that the standing times for the coatings were 3 h, 5 h, 10 h and 12 h, respectively. The mixture of RNase A and DMSO was adopted as an analysis object of CE, and results of CE were used to investigate the performance of the four BSA-coated capillaries. When the standing time of the coating was 2 h, the theoretical plate number of RNase A was only 8500 N/m; when the standing time increased to 5 h, the theoretical plate number of RNase A increased to 9.4 x 104 N/m; when the standing time increased to 10 h, the theoretical plate number of RNase A increased to 1.0 x 105 N/m; and when the standing time increased to 12 h, the theoretical plate number of

RNase A increased to 1.02 x 105 N/m. When the standing time increased to from 3 h to 5 h, the theoretical plate number of RNase A increased nearly 12 times, indicating that the standing time of 3 h was far from enough to form a prominent BSA coating. However, when the standing time increased from 5 h to 10 h and 12 h, although a long standing time interval was elapsed, the theoretical plate number increased very little, indicating that the BSA coating had exhibited an excellent adsorption state when standing for 10 h. Therefore, the optimal standing time for the BSA coating was finally determined as 10 h. (2) Stability evaluation of the inner wall coating of the differential microfluidic plate channel The highly-sensitive detection of genetically-modified proteins has a hierarchical feature, that is, the previous processing results are the basis for the latter. In order to eliminate the error accumulation effect and determine whether the BSA coating meets experimental requirements, the stability of the inner wall coating of the microfluidic plate channel must be evaluated. The present disclosure adopted EOF and theoretical plate number of RNase A for evaluation. Calculation formula of EOF:

where, represents the migration time of DMSO, represents the diameter of the inner wall of the terahertz microfluidic plate channel (cm), !represents the total length of the terahertz microfluidic plate channel (cm), and a represents the separation voltage (V). Electrophoresis was conducted continuously for 48 h using the coated microfluidic plate channel, and the EOF and the theoretical plate number of RNase A were recorded every 12 h.

Relative standard deviation (RSD) was calculated for EOF and theoretical plate number of RNase A according to recorded data. Only an RSD lower than a specified value (such as, 10%) can prove that the inner wall coating of the microfluidic plate channel has high stability. Experimental results were shown in the table. It can be seen from Table 1 that, at the initial stage, the EOF of the coated capillary was 2.56± 0.04 (x 10-4 cm2 /V/s), and the theoretical plate number of RNase A is 1.34 ±0.03 (x 105); and after electrophoresis was continuously conducted for 48 h using the coated capillary, the EOF became 2.65 ±0.04 (x 10-4cm 2/V/s), and the theoretical plate number of RNase A became 1.25 ±1.61 (x 105). In addition, after 48 h of continuous electrophoresis, the RSD values of EOF and theoretical plate number were 4.14% and 9.14%, respectively. These results are sufficient to prove that the coating has a high stability. Table 1 EOF and RNase A theoretical plate number of the coated capillary

Time interval (X 10 4CM 2 / Theoretical plate number for CE (h) FOF cms) of RNase A (X 10 5N/m) 0 2.65 ± 0.04 1.34 ± 0.03

12 2.43 ± 1.06 1.32 ± 0.02

24 2.67 ± 0.02 1.30 ± 1.17

36 2.71 ± 0.06 1.06 ± 1.24

48 2.65 ± 0.04 1.25 ± 1.61

RSD (%) 4.14 9.14

Selection of an appropriate operating frequency band Achieving the extraction of the terahertz characteristic spectrum of a genetically-modified target protein at high signal-to-noise ratio (SNR) is of great significance for selecting effective processing methods and improving processing accuracy. Therefore, it is necessary to screen the operating frequency bands of the terahertz differential microfluidic plate channel to prevent other factors from interfering with the extraction of the characteristic spectrum. By optimizing the spacing between the two plates, coupling occurs between the two plates within a specified frequency range, and the number of resonant frequencies at which overlapping occurs between the two plates is unique. According to the analysis, only the overlapping of resonant frequencies can meet the condition for eliminating non-sensing drift interference. When an appropriate spacing between plates is selected, the resonant frequencies of the two plates will overlap, so that the sensing operating frequency can be individually screened out for observation. With the THz-TDS system as a measurement platform, the present disclosure adopted the transmission line theory to calculate the resonance frequency of the microfluidic plate channel, and plotted the frequency response characteristic curve. In order to test the usability of the terahertz biosensor, before the biosensor was used to measure a biochemical liquid sample, the liquid (water) playing a very important role in biological and chemical activities was measured in the terahertz frequency band. Experimental results showed that the absorption coefficient and refractive index curves of water in the range of 0.3 THz to 1.1 THz were well consistent with the results measured using liquid sample cells made of different materials, as shown in FIG. 3. When 0.3 THz to 1.1 THz was adopted as the operating frequency band, no obvious absorption peak was observed for water, and a high transmittance of terahertz waves was exhibited (above 50%, up to 85%). Therefore, this frequency band was selected as the effective frequency range of the terahertz sensor according to the present disclosure. Capture and release of a target protein The present disclosure realizes the capture and release of the cis-dihydroxy biomolecule adenosine by adjusting the temperature, and the principle is shown in FIG. 7. When the temperature is lower than the lower critical solution temperature (LCST), the grafted P(NIPAAm-co-VPBA) polymer is in a hydrophilic state, and the P chain stretches out so that the binding sites (borate groups) for a target substance on the chain are exposed, which will capture cis-dihydroxy biomolecules in a sample. When the temperature is increased to above the LCST, the grafted polymer is in a hydrophobic state, and the P chain changes from stretching to coiling so that the captured cis-dihydroxy biomolecules fall off. The capture and release of the cis-dihydroxy biomolecule adenosine is achieved by adjusting the temperature in the above manner. The hydrophilic and hydrophobic properties (wettability) of the P polymer can be indirectly characterized by measuring the contact angle of a liquid on the solid. The contact angles of a liquid on the glass substrate with thermoresponsive boronate affinity at different temperatures are shown in FIG. 8. It can be observed from FIG. 8 that, when the temperature of the substrate is maintained at 4°C, the measured contact angle is 30.6°; when the temperature rises to 25°C, the contact angle of water increases to 53.1°, indicating that the hydrophilicity of the surface decreases when the temperature rises from 4°C to 25°C; and when the temperature rises to 55°C, the contact angle with the P-grafted glass substrate rapidly increases to 93.3°. It shows that, when the temperature is 55°C, the surface of the P-grafted glass substrate is completely in a hydrophobic state. Since the protein starts heat denaturation at about 60°C, a target substance is captured at 4°C to 55°C and released at a temperature < 4°C. A sample detection method using the terahertz biosensor provided by the present disclosure may preferably include the following steps:

(1) A Bt protein liquid sample (buffer: hydroxyethylpiperazine ethanesulfonic acid (HEPES)) with a concentration of 0.1 mg/ML was injected into a chip channel by a micro-injection pump and kept at 2°C for 3 min. (2) A THz-TDS system with a terahertz spot size of 2 cm was used to irradiate the microfluidic plate channel to obtain the TDS data of the sample. (3) In order to avoid random errors, the TDS data were measured 5 times, with a time interval of 1 min, and the average value was calculated. (4) The microfluidic chip was placed on a 55°C hot plate for 3 min, then the channel was rinsed twice with an HEPES buffer (2.383 g HEPES was dissolved in 900 mL of ultrapure water, then pH was adjusted to 9.8 using NaOH, and a resulting solution was diluted to 1 L), and the entire channel was fully filled with the HEPES buffer to be ready for the next measurement. Experimental samples: Table 2 List of test experimental samples No. Name Type Specification Supplier

Lu Mianyan Transgenic Bt insect-resistant Shandong Luyin Cotton

No. 36 conventional varieties Industry

Xinqiu Transgenic Bt insect-resistant An HEPES mixed solution 2 Shandong Xinqiu Seed Industry K638 cotton varieties with a concentration of 0.1

Xin Non-transgenic early- and mg/ML Cotton Research Institute,

3 Luzhong middle-maturing conventional Chinese Academy of

No. 6 varieties Agricultural Sciences

Test results were shown in FIG. 9. It can be seen from the experimental results that the cotton varieties with the Bt target protein can be effectively detected by the test method of the present disclosure, which can achieve a detection rate of 100% in terms of the 5 times of detection for each cotton variety. The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

Claims

What is claimed is: 1. A terahertz biosensor, comprising a terahertz differential microfluidic plate channel; wherein, the terahertz differential microfluidic plate channel comprises an inlet channel, a first microfluidic plate, a second microfluidic plate and an outlet channel that are successively connected; the first microfluidic plate and the second microfluidic plate are connected via a microchannel; the terahertz differential microfluidic plate channel adopts quartz glass as a substrate and polydimethylsiloxane (PDMS) as a cover film; and the surface of the quartz glass is grafted with P(NIPAAm-co-VPBA) having thermoresponsive boronate affinity.
2. The terahertz biosensor according to claim 1, wherein, the terahertz differential microfluidic plate channel has a depth of 40 m to 60 m; the first and second microfluidic plates have a spacing of 20 m to 40 m; and the microchannel has a width of 10 m to 100 [m.
3. The terahertz biosensor according to claim 1, wherein, the inner wall of the terahertz differential microfluidic plate channel is provided with a bovine serum albumin (BSA) coating; a coating agent used for the BSA coating has a BSA concentration of 1 mg/mL to 2 mg/mL; and the coating agent has a pH of 4.0 to 5.0.
4. A method for preparing the terahertz biosensor according to any one of claims 1 to 3, comprising the following steps: 1) grafting P(NIPAAm-co-VPBA) on the surface of a quartz glass substrate by SI-ATRP reaction to obtain a glass substrate with thermoresponsive boronate affinity; 2) subjecting an embossed mask with a differential microfluidic plate channel structure to photoetching and photocuring using SU-8 photoresist under the action of ultraviolet (UV), and rinsing to obtain a mask with a photoresist layer; 3) bonding the mask with a photoresist layer and the glass substrate with thermoresponsive boronate affinity, and removing the mask to obtain a substrate of the terahertz differential microfluidic plate channel; and 4) bonding the substrate of the terahertz differential microfluidic plate channel with an SU-8 photoresist cover sheet to obtain the terahertz differential microfluidic plate channel, wherein, the SU-8 photoresist cover sheet has an inlet channel and an outlet channel.
5. The preparation method according to claim 4, wherein, the method further comprises: injecting the coating agent into the terahertz differential microfluidic plate channel for coating, and standing a resulting coating for 5 h to 10 h; wherein, the photoetching and photocuring is conducted for 30 s to 35 s.

1 /7 28 Jan 2021

S2 S1 S201 S101 S1 03 2021100539

S104 S202 S203 S204

S102 S106

S105 S205 S107

S108 S109 S110 S111

FIG. 1

Quartz glass Quartz glass PDMS Microfluidic channel inlet Microfluidic outlet 2 /7

Microfluidic

FIG. 2 plate 1 plate 2 PDMS

THz radiation

Absorption coefficient/a.u. 3 /7

FIG. 4 FIG. 3 Frequency/THz

Refractive index

4 /7 28 Jan 2021

OH O OH O Si (CH2)3 NH2 OH O

2-Bromoisobutyryl bromide

O CH3 O O Si (CH2)3 NH C C Br O 2021100539

CH3 NIPAAm+VPBA

O CH3 O O Si (CH2)3 NH C C CH2 CH CH2 CH Br O x y

CH3 C O

NH

CH B

CH3 CH3 HO OH

FIG. 5

5 /7

Chrome plate Chrome plate 28 Jan 2021

Spread a Spread a photoresist photoresist SU8 photoresist SU8 photoresist

Mask with a Mask with a microfluidic plate cover sheet channel structure structure (inlet and outlet) Mask Mask 2021100539

UV curing UV curing

Rinse away Rinse away uncured uncured photoresist photoresist

Bonding Bonding

Quartz glass substrate with Quartz glass substrate with thermoresponsive boronate affinity thermoresponsive boronate affinity

Remove the Remove the mask mask

Quartz glass substrate with Quartz glass substrate with thermoresponsive boronate affinity thermoresponsive boronate affinity Final structure

FIG. 6

6 /7

FIG. 7

7 /7 28 Jan 2021 2021100539

FIG. 8

Reference signal Lu Mianyan No. 36 Xinqiu K638 Xin Luzhong No. 6

FIG. 9