Arsenic ion synthetic biosensor based on cell-free system and application thereof

Abstract

The application discloses a paper-based arsenic ion synthetic biosensor based on a cell-free system and application thereof, and comprises a cell-free arsenic ion sensing module and a semi-quantitative paper-based detection chip. The cell-free arsenic ion sensing module is composed of a synthetic arsenate reduction module, an arsenic ion specificity sensing module, a high-gain transcription amplification module and a visual reporting module connected in sequence. The synthetic arsenate reduction module converts pentavalent arsenic into trivalent arsenic; the arsenic ion specificity sensing module recognizes trivalent arsenic and removes transcription inhibition; the high-gain transcription amplification module amplifies the signal through ecf11 cascade; and the visual reporting module outputs a colorimetric or fluorescent signal. The application integrates an engineered promoter and an ecf11 cascade amplification system in a cell-free system, greatly improves sensitivity and dynamic range and keeps low leakage. The paper-based chip can present different pattern arrangements according to arsenic concentration and is easy to read. The application provides an environmentally friendly, portable and easy-to-read innovative method for monitoring arsenic pollution in drinking water.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, specifically relating to an arsenic ion synthesis biosensor based on a cell-free system and its application, which is suitable for rapid on-site detection of arsenic pollution in drinking water in resource-scarce environments. Background Technology

[0002] Currently, both the World Health Organization's Guidelines for Drinking-water Quality and my country's GB 5749-2022 Standards for Drinking Water Quality strictly set the limit for arsenic in drinking water at 10 ppb. Since arsenic is odorless and tasteless and cannot be detected by sensory means, developing a low-cost, environmentally friendly, easy-to-operate, and on-site-deployable rapid detection technology for arsenic pollution is of great significance for ensuring drinking water safety.

[0003] Existing arsenic detection technologies in water bodies are mainly divided into two categories: standard laboratory detection methods and rapid on-site detection methods. Both have insurmountable limitations in application. Standard laboratory methods, represented by inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS), offer high detection accuracy and good repeatability, but the instruments are expensive, require professional personnel for operation, involve complex sample pretreatment, have long detection cycles, and are costly, making them unsuitable for on-site deployment and large-scale screening.

[0004] Existing rapid on-site detection technologies also have core drawbacks: chemical colorimetric methods (represented by the Gutzeit method) are simple to operate, but they produce highly toxic arsine gas, and the detection limits are generally higher than 50 ppb, which cannot meet the detection requirements of the 10 ppb safety limit. Moreover, the colorimetric results are easily affected by environmental interference and have poor repeatability. Electrochemical detection methods have high sensitivity, but the electrodes are easily affected by coexisting metal ions in water, have poor stability in complex matrices, require regular calibration and maintenance, and have high equipment costs, making it difficult to promote on a large scale. Whole-cell biosensors have high specificity and low preparation costs, but they pose biosafety risks from genetically modified microorganisms, require cold chain storage and transportation, are easily inactivated at room temperature, have detection cycles of up to 4-6 hours, and have serious cross-reaction problems.

[0005] Cell-free biosensors driven by synthetic biology represent an important direction in the development of biosensors. They achieve target detection by extracting the contents of *E. coli*, adding energy buffer and DNA template, and reconstructing the gene transcription and translation system in vitro. Compared to whole-cell biosensors, cell-free systems eliminate the cell membrane barrier, theoretically resulting in superior detection sensitivity; they eliminate the involvement of living cells and the risk of environmental release from genetically modified microorganisms; they require no bacterial proliferation, leading to faster response times; and they can be formulated through freeze-drying, allowing for room-temperature storage and transportation without the need for a cold chain, making them suitable for field applications in resource-scarce regions.

[0006] However, the cell-free arsenic ion biosensors reported so far still have a series of key technical bottlenecks that cannot meet the needs of practical field applications: First, insufficient detection sensitivity. Most sensors have no signal or weak response at the 10 ppb safety limit, which cannot meet the needs of practical applications. Existing signal amplification technologies often sacrifice the dynamic range of the sensor or rely on expensive reagents, making it difficult to apply on a large scale. Second, narrow detection spectrum. The core recognition element can only specifically recognize As(III), and has a weak response to As(V), which is widely coexisting in natural water bodies, only at high concentrations, which can easily lead to missed detections. Third, poor practicality of signal output. Most of them use fluorescence signal output, rely on expensive detection equipment, and cannot be visualized with the naked eye. A few colorimetric sensors can only provide binary colorimetric results, which cannot realize the graded determination of arsenic concentration, and non-professionals are prone to misjudgment. Fourth, insufficient field adaptability. The storage period after freeze-drying is short, the activity is severely lost during room temperature transportation, the anti-interference ability in complex water matrices is weak, and the detection stability in non-laboratory environments is poor.

[0007] In summary, existing water arsenic detection technologies cannot simultaneously meet the core requirements of high sensitivity, broad detection spectrum, low cost, ease of operation, high biosafety, and stable on-site deployment. They are particularly ill-suited for large-scale on-site screening of arsenic contamination in drinking water in resource-scarce regions. Therefore, developing a cell-free paper-based arsenic biosensor capable of overcoming these technological bottlenecks is a pressing technical challenge in this field. Summary of the Invention

[0008] To address the core shortcomings of existing arsenic detection technologies in water bodies, such as insufficient sensitivity, narrow detection spectrum, poor on-site adaptability, high biosafety risks, and difficulty in interpreting results, this invention aims to provide a paper-based arsenic ion biosensor based on a cell-free synthetic biology system, along with the simultaneous development of a corresponding sensing system, on-site detection system, preparation method, and application scheme. This invention enables simultaneous, highly sensitive detection of trivalent arsenic (As(III)) and pentavalent arsenic (As(V)), achieving a stable signal response within the WHO-specified 10 ppb safety limit for drinking water arsenic. It also boasts advantages such as naked-eye visualization for grading and determination, stability during room temperature storage and transportation, low cost, ease of operation, and no biosafety risks associated with live bacteria. Therefore, it is perfectly suited for large-scale on-site screening and rapid detection of arsenic contamination in drinking water in resource-scarce areas.

[0009] To achieve the above objectives, the specific technical solution of this invention is as follows, which includes two core components: a cell-free arsenic ion biosensing system and a visual paper-based colorimetric sensor array, along with standardized preparation and on-site detection methods.

[0010] In a first aspect, the present invention provides an arsenic ion synthesis biosensor based on a cell-free system, comprising a cell-free arsenic ion sensing module, wherein the cell-free arsenic ion sensing module is composed of the following functional units connected sequentially in the order of signal transmission: (a) A synthesis arsenic acid reduction module, used to directionally convert pentavalent arsenic into trivalent arsenic; (b) An arsenic ion sensing module comprising an arsenic-responsive promoter and an arsenic-responsive transcription factor, wherein the arsenic-responsive transcription factor undergoes a conformational change in the presence of trivalent arsenic, thereby relieving transcriptional repression of the arsenic-responsive promoter; (c) Signal gain processing module, containing signals from Vibrio parahaemolyticus ( Vibrio parahaemolyticus The extracellular functional σ factor ecf11 and its homologous promoter P ecf11 The output of the arsenic-responsive promoter is concatenated with the coding sequence of ecf11, wherein the P ecf11 Used to drive downstream gene expression; (d) A reporter module, containing a reporting element, for outputting a detectable signal; All of the above modules are integrated into a cell-free expression system.

[0011] Specifically, a cell-free system-based arsenic ion synthesis biosensor system: The sensing system of this invention is based on modular synthetic biology design, and its core consists of four parts: a synthetic arsenic acid reduction module, an arsenic ion sensing module, a signal gain processing module, and a reporter module. It possesses ultra-sensitive arsenic ion detection performance. Figure 1 ).

[0012] 1. Arsenic ion sensing module This module is the core of arsenic ion recognition, driven by a constitutive promoter that expresses the arsenic-specific repressor protein ArsR (i.e., an arsenic-responsive transcription factor), and a rationally engineered arsenic-responsive promoter P. arsRD constitute.

[0013] The ArsR protein is an arsenic-specific allosteric transcriptional repressor protein. In the absence of arsenic, it can specifically bind to the binding site of the arsenic-responsive promoter and inhibit the transcription of downstream genes. In the presence of As(III), it can specifically bind to As(III) and undergo a conformational change, dissociating from the promoter, relieving transcriptional repression, and initiating the expression of downstream functional genes.

[0014] The P arsRD The promoter is a hybrid engineered promoter, developed by modifying wild-type P arsRD A mutation in the -10 / -35 core functional region of the promoter, along with the addition of an additional ArsR binding site (ABS) downstream of the transcription start site, achieves transcriptional repression. Under 50 ppb arsenic induction, it achieves a 32.3-fold dynamic response range, a 7.9-fold increase compared to the wild-type promoter, while significantly reducing background leakage. This solves the core problems of high background leakage and low response folds in traditional arsenic-responsive promoters. Figure 2 ).

[0015] 2. Arsenic acid reduction module This module is the core functional unit for expanding the arsenic detection spectrum. It is used to solve the technical bottleneck of traditional arsenic sensors that can only identify As(III) and cannot effectively detect As(V) which is widely coexisting in natural water bodies, and to achieve simultaneous equivalent detection of As(III) and As(V).

[0016] This module is designed based on the in vitro reconstruction of the E. coli arsenic detoxification metabolic pathway. Its core consists of ArsC, an arsenate reductase expressed under a constitutive promoter, supplemented by glutathione (GSH), a naturally occurring electron donor in the reaction system. Its mechanism of action is as follows: ArsC specifically binds to As(V) in water, using GSH as an electron donor, efficiently completing the enzymatic reduction reaction in a cell-free system. This converts As(V) into As(III), which can be recognized by the arsenic-specific sensing module, thus enabling the sensing system to produce a recognition response to As(V) that is completely equivalent to that to As(III).

[0017] Performance verification shows that this module can enhance the As(V)-induced signal output to a level indistinguishable from that of As(III), without negatively impacting the As(III) detection performance of the sensing system, and is fully compatible with downstream transcription amplification modules. This module overcomes the limitation that the ArsR protein can only specifically recognize As(III) ligands, achieving full coverage detection of the two main inorganic arsenic forms in natural groundwater without modifying the core recognition element. This fundamentally solves the core defects of traditional arsenic sensors, such as low sensitivity and easy missed detection of As(V). Figure 3 ).

[0018] 3. Signal Gain Processing Module This module is the core functional unit for improving detection sensitivity. It addresses the core pain points of traditional cell-free sensors, such as weak signal response and low sensitivity under the WHO-specified safety limit of 10 ppb. It achieves high-magnification amplification of low-concentration arsenic signals while maintaining extremely low background signal.

[0019] This module is constructed based on the extracellular functional σ factor ecf11_987 of Vibrio parahaemolyticus, and adopts a cascade transcription amplification design: the arsenic-responsive promoter P... arsRD The output is concatenated with the ECF11 coding sequence, and then passed through the ECF11 specific homologous promoter P. ecf11 It drives downstream reporter module expression. Its amplification mechanism is as follows: the weak transcriptional signal triggered by arsenic is amplified by P... arsRD The promoter drives the expression of ecf11, which can bind with high affinity to its homologous promoter P. ecf11 It recruits RNA polymerase to initiate high-intensity transcription of downstream genes, thereby linearly amplifying the initial weak signal at a high rate.

[0020] Performance verification shows that this module can increase the signal response at a 10 ppb arsenic concentration from 3 to 13 times, and achieve a 19-fold scaling of the linear dynamic range, reducing the detection limit of the sensing system to as low as 1 ppb, which is 10 times lower than the detection limit of existing cell-free arsenic sensors. It is also fully compatible with the upstream arsenic-specific sensing module and the synthetic arsenate reduction module, exhibiting stable signal amplification for both As(III) and As(V), thus overcoming the technical shortcomings of traditional signal amplification techniques that easily sacrifice dynamic range and rely on expensive reagents. Figure 4 ).

[0021] 4. Reporting module This module serves as the signal output terminal of the sensing system, converting transcribed and amplified biological signals into colorimetric signals that can be read by the naked eye. It eliminates the need for specialized detection equipment and is suitable for the result interpretation needs of non-professional personnel on-site.

[0022] This module employs a LacZα / ω complementary chromogenic system. The output of a high-gain transcription amplification module drives the expression of the LacZα peptide, which assembles in vitro with the pre-expressed LacZω fragment to form a functional tetramer protein with complete β-galactosidase activity. This tetramer catalyzes the hydrolysis of the chromogenic substrate chlorophenol red-β-D-galactopyranoside (CPRG), changing the reaction system from yellow to purple, thus achieving a visually perceptible signal output. Furthermore, by adjusting the DNA template concentration in the cell-free system, the sensor's response threshold can be precisely controlled to adapt to the detection requirements of different arsenic concentration levels, providing a standardized basis for subsequent semi-quantitative grading determination using paper-based arrays. Figure 5 ).

[0023] 5. Cell-free reaction basic system All four functional modules mentioned above are integrated into the Escherichia coli cell-free expression system (CFS). The basic system includes E. coli S30 cell extract, energy buffer, nuclease inhibitor GamS, amino acid substrate and coenzyme components, which can complete the entire transcription-translation process in vitro, realize the controllable expression of sensing elements and cascade signal output, without the participation of living cells, and the biosafety is controllable throughout the process.

[0024] Preferably, the arsenic-responsive promoter is a P nucleotide sequence as shown in SEQ ID No. 1. arsRD ; The arsenic-responsive transcription factor is ArsR, with a nucleotide sequence as shown in SEQ ID No. 2; The synthetic arsenate reduction module contains an arsenate reductase ArsC with the nucleotide sequence shown in SEQ ID No. 3; The nucleotide sequence of the ecf11 σ factor is shown in SEQ ID No. 4, and the Pecf11 The nucleotide sequence is shown in SEQ ID No. 5; The reporter module comprises a complementary colorimetric system of LacZα and LacZω nucleotide sequences as shown in SEQ ID No. 6 and SEQ ID No. 7, or a fluorescent protein mScarlet with a nucleotide sequence as shown in SEQ ID No. 8.

[0025] Furthermore, the arsenic ion synthesis biosensor based on the cell-free system also includes a semi-quantitative paper-based detection chip. The chip is formed by treating filter paper with a hydrophobic barrier to create five linearly arranged dot-matrix reaction zones, which are, in order, a negative control zone, a first arsenic ion sensing system zone, a second arsenic ion sensing system zone, a third arsenic ion sensing system zone, and a positive control zone. The arsenic ion sensitivity of the first, second, and third arsenic ion sensing system zones increases sequentially, corresponding to arsenic ion concentrations of 100 ppb, 50 ppb, and 10 ppb, respectively. The color development mode of the chip is such that the number of reaction zones that change from yellow to purple is positively correlated with the concentration of arsenic ions in the water sample being tested.

[0026] The five wells are arranged linearly, from left to right: negative control, arsenic ion sensing system 1 (lowest sensitivity), arsenic ion sensing system 2 (medium sensitivity), arsenic ion sensing system 3 (highest sensitivity), and positive control. The designed paper-based detection chip can present a very intuitive, battery-like response pattern for water samples with different arsenic ion concentrations: the more wells that change from yellow to purple, the higher the concentration of arsenic ions in the water sample.

[0027] Specifically, a visualized paper-based colorimetric sensor array: Based on the aforementioned sensing system, this invention develops a paper-based microfluidic sensor array with a "traffic light" style graded output, enabling naked-eye visual graded determination of arsenic concentration. The core structure and design are as follows: Array Substrate and Structure: Cytiva medium-speed qualitative filter paper was used as the substrate, and a hydrophobic barrier was prepared by wax printing to form five independent, non-cross-contamination circular reaction zones. Figure 6 In the diagram, a) is arranged linearly as a negative control area (N), three sensors with different sensitivities (S1~S3), and a positive control area (P).

[0028] The partitioning function is designed as follows: 1. The negative control area (N) contains only the cell-free basic system and the chromogenic substrate CPRG, without the sensor-related DNA template, and is used to subtract background interference from the environment and water sample matrix; 2. Low concentration response zone (S1): Low-sensitivity arsenic ion sensor freeze-drying system, responding to 100 ppb arsenic ion water sample, corresponding to severe arsenic ion pollution; 3. Medium concentration response zone (S2): A freeze-dried system for a medium-sensitivity arsenic ion sensor, responding to a 50 ppb arsenic ion water sample, corresponding to the drinking water safety limit in areas with severe arsenic pollution; 4. High concentration response zone (S3): High-sensitivity arsenic ion sensor freeze-drying system, responding to 10 ppb arsenic ion water samples, corresponding to the WHO-specified drinking water safety limit; 5. Positive control area (P): Cell-free system containing constitutive promoter-driven LacZα expression, used to verify the activity and effectiveness of the cell-free system and exclude false negative results.

[0029] After quantitatively freeze-drying the cell-free sensing system corresponding to the above partitions to the paper-based reaction zone, the response fully met the design expectations, and the response results for As(III) and As(V) were the same. Figure 6 (b) After freeze-drying, when vacuum-sealed with a desiccant and stored in a light-proof container at 4°C, it remains stable for over 60 days, and there is no significant loss of activity during normal temperature transportation. Figure 6 (d in the original text). Meanwhile, this sensor does not respond to most common metal ions found in water, demonstrating excellent specificity for arsenic ions (d in the original text). Figure 6 (c) Since the chromogenic substrate CPRG changes from yellow to purple through a catalytic reaction, the green channel shows the most significant change among the three RGB channels; therefore, only the green channel image is displayed.

[0030] Furthermore, the semi-quantitative paper-based detection chip is prepared using an ice bucket freeze-drying method: the paper-based detection chip is placed in an aluminum cylindrical tube, pre-cooled at -80°C, and then freeze-dried together with the aluminum tube; bovine serum albumin is added before freeze-drying to prevent non-specific adsorption, thereby improving the expression efficiency of cell-free systems. Simultaneously, BSA can adsorb some toxic substances in natural water samples, increasing the robustness of the cell-free system.

[0031] Furthermore, the bacterial strain used to prepare the cell-free expression system is BL21 Star ΔlacZ, which is a knockout strain. lacZ The gene was used in *E. coli* BL21 Star (DE3) to avoid non-specific catalysis of the CPRG chromogenic substrate; lacZ The gene sequence is shown in SEQ ID No. 12 and is used for knockout. lacZ The gene sequence of the DNA fragment of the gene is shown in SEQ ID No. 13.

[0032] All the genes used in the sensing module are linear DNA fragments, each of which contains transcription terminator elements on both sides, forming a secondary structure that slows down the degradation rate of linear DNA.

[0033] Furthermore, the constitutive promoters used to express LacZω, ArsR, and ArsC are selected from SJM901, J23101*, and J23101, respectively, and their nucleotide sequences are shown in SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 11, respectively. In the positive control, the J23101 promoter was used to express mScarlet or LacZα.

[0034] Furthermore, the cell-free arsenic ion sensing module was prepared using a pre-expression method: First, the DNA circuit containing the arsenic-responsive transcription factor ArsR was added to a cell-free system and pre-reacted at 37°C for 30 minutes; then, reporter element DNA and chromogenic substrate were added for colorimetric reaction. The ArsC and ArsR circuits in the synthetic arsenic acid reduction module are added simultaneously during the pre-expression stage to achieve simultaneous equivalent detection of pentavalent and trivalent arsenic.

[0035] Furthermore, by adjusting the concentration of each DNA template in the cell-free arsenic ion sensing module, the sensitivity and dynamic response range of the sensor to arsenic ions are changed, forming a sensing system with three different sensitivity gradients.

[0036] On the other hand, the present invention also provides the application of the aforementioned arsenic ion synthesis biosensor in the on-site detection of arsenic pollution in drinking water.

[0037] When applying, the following steps are included: (1) Take the drinking water sample to be tested, which does not require pretreatment or only requires static sedimentation treatment; (2) Add the drinking water sample to be tested to each reaction zone of the semi-quantitative paper-based detection chip to rehydrate the freeze-dried cell-free arsenic ion sensing module. (3) Incubate the paper-based detection chip at 30°C for 2 hours; (4) Semi-quantitative interpretation can be made by observing the color changes of each reaction area with the naked eye, or by taking pictures with a smartphone for analysis.

[0038] Finally, the present invention also provides a method for preparing an arsenic ion synthesis biosensor based on a cell-free system, comprising: (a) Construction of a cell-free arsenic ion sensing module: The linear DNA templates for synthesizing the arsenic acid reduction module, arsenic ion sensing module, signal gain processing module, and reporter module are mixed with a cell-free expression system, wherein the DNA templates contain the sequence elements shown in SEQ ID No. 1 to SEQ ID No. 11; (b) Preparation of semi-quantitative paper-based detection chip: A hydrophobic barrier was prepared on filter paper using paraffin printing technology to form five linearly arranged dot array reaction areas, which correspond to the negative control, the arsenic ion sensing system with three sensitivity gradients and the positive control, respectively. (c) Spot the cell-free sensing system obtained in step (a) onto the corresponding reaction area of ​​the chip in step (b), and freeze-dry it using the ice bucket low-temperature freeze-drying method to obtain a paper-based freeze-dried biosensor, namely the arsenic ion synthesis biosensor.

[0039] The beneficial effects of this invention are: 1. Ultra-high detection sensitivity and wide dynamic range: Through rational engineered promoter and synergistic optimization of cascaded transcription amplifier, a detection limit of 1 ppb for arsenic is achieved, with a signal response of more than 12 times at the WHO safety limit of 10 ppb and a dynamic range of 32.3 times at 50 ppb, which is far superior to the cell-free arsenic biosensors reported to date. 2. Simultaneous detection of all valence states of arsenic: By reconstructing the arsenate reduction pathway in vitro, the detection of the two main toxic arsenic forms, As(III) and As(V), with equal efficiency was achieved for the first time in a cell-free system, solving the core problem that existing sensors can only detect a single valence state of arsenic and are prone to false negative results; 3. Excellent field adaptability and stability: The paper-based freeze-dried sensor array can be stably stored at 4℃ for more than 2 months. Its performance does not significantly degrade after transportation at room temperature. The cost of a single test is only US$0.4. No complicated sample pretreatment is required. The test can be completed within 2 hours. The results are directly interpretable by the naked eye through "traffic light" style color development, which is fully adapted to the field testing needs of low-resource areas. 4. Strong specificity and anti-interference ability: The sensor has high specificity for arsenic and no cross-response to more than ten metal ions commonly found in the aquatic environment, such as zinc, mercury, manganese, copper, lead, cadmium, iron, nickel, calcium, and cobalt. It can be directly used for the detection of complex matrix water samples such as untreated groundwater, and the detection rate of polluted water samples exceeding WHO safety limits is as high as 100%. 5. High biosafety and environmental friendliness: It adopts a cell-free in vitro expression system, eliminating the biosafety risks associated with genetically modified live microorganisms. The detection process does not use toxic reagents or generate harmful waste, making it safer in terms of environment and operation compared to traditional detection technologies such as the Gutzländer method. 6. Modularity and scalability: The sensing system adopts a modular design. The core valence state conversion module and signal amplification module can be adapted to sensing modules with different specificities, and can be quickly expanded to the on-site detection of other water environment pollutants, with strong versatility and application prospects. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the principle of the present invention; wherein, a is a schematic diagram of the gene circuit of the ultrasensitive arsenic ion synthesis biosensor, and b is a conceptual diagram of the preparation and application of the paper-based arsenic ion sensor.

[0041] Figure 2 The following describes the engineering modification and characterization of the arsenic ion-responsive promoter; where a represents the promoter engineering modification strategy; and b represents a comparison of the arsenic ion response characterization results between the optimized promoter and the wild-type promoter.

[0042] Figure 3 Characterization of the arsenic acid reduction module; where a is the arsenic acid reduction pathway and circuit diagram; b is the circuit diagram for adding arsenic acid reductase expression to the cell-free sensing system ( arsC ), glutathione (GSH) and glutreonin expression pathways ( grx The response results to pentavalent arsenic ions.

[0043] Figure 4 The amplifier module (signal gain processing module) is constructed and characterized; where a is the schematic diagram of the amplifier circuit design; b is a comparison of the dose response curve of the arsenic ion sensor of the coupled amplification circuit to trivalent arsenic ions and the response level of the 10 ppb safety limit; c is a comparison of the dose response curve of the arsenic ion sensor of the coupled amplification circuit to pentavalent arsenic ions and the response level of the 10 ppb safety limit.

[0044] Figure 5 The colorimetric report module for the arsenic ion sensor output module is constructed; where a is a schematic diagram of the sensing circuit; b is a photograph of the sensing array and a heat map of the B / G signal values.

[0045] Figure 6 Design and characterization of a paper-based arsenic ion sensor array; wherein, a is a conceptual diagram of an easily readable paper-based arsenic ion sensor array; b is a colorimetric image of the arsenic ion sensor array for several key concentrations of trivalent and pentavalent arsenic ions; c is the specific characterization of the paper-based arsenic ion sensor; d is the verification of the shelf life of the paper-based sensor.

[0046] Figure 7 This is a circuit diagram of the plasmid used in this invention.

[0047] Figure 8This is a schematic diagram of the fabrication and packaging of a paper-based colorimetric sensor; where a is the hydrophobic pattern design of the paper-based sensor; and b is the 3D printing magnetic support design of the paper-based sensor.

[0048] Figure 9 The results of the arsenic ion synthesis biosensor of this invention detecting actual samples are shown.

[0049] The scatter plots represent parallel samples. ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0050] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0051] Example 1: Construction of a cell-free arsenic synthesis biosensor 1. Construction of cell-free expression strain: Based on Escherichia coli BL21 Star (DE3) (Qingke Biotechnology, TSC-E06), Escherichia coli BL21 Star (DE3) Δ was constructed through λ-Red gene recombination. lacZ The steps are as follows: First, prepare a mixture containing... lacZ DNA fragments of the upstream and downstream homologous arms of the gene and the chloramphenicol resistance gene expression cassette (SEQ ID No. 13); subsequently, the λ-Red homologous recombinant plasmid pSim6 (Newp Biotechnology, product number: V012110) was transformed into Escherichia coli BL21Star (DE3) competent cells; the positive strain was cultured to OD 600 When the value was 0.4-0.6, λ-Red recombinase was induced to express by heat shock at 42℃ to prepare electroporation competent cells; then, the previously prepared DNA fragment was electroporated into these competent cells, and positive clones were obtained by chloramphenicol resistance selection; finally, PCR amplification and gene sequencing confirmed the presence of λ-Red recombinase in the strain genome. lacZ The gene (SEQ ID No. 12) was successfully knocked out, resulting in the target cell-free expression engineered strain BL21 Star (DE3)Δ. lacZ .

[0052] 2. Preparation of cell-free system: The self-made cell-free system mainly consists of bacterial lysate and energy buffer.

[0053] The steps for preparing bacterial lysates are as follows: Pick Escherichia coli strain BL21 Star (DE3) Δ lacZ Single clones were grown overnight in 2×YTPG medium (OD) 600~10), dilute 200-fold and transfer to 200 mL of 2×YTPG medium, incubate in a 500 mL shake flask with baffle at 37°C, until approximately 4 hours later, at which point the OD200 reaches mid-log phase. 600 Cells were collected (value 3.5-4) and washed twice with pre-chilled Buffer A (10 mM Tris-acetate buffer, 14 mM magnesium glutamate, 60 mM potassium glutamate), blotted dry, and stored at -80°C. The stored bacteria were resuspended in Buffer A containing 2 mM DTT, with the volume of liquid added equal to the mass of the bacteria. Cells were sonicated at a work / interval of 5 s / 10 s for a total of 2 min, with a phase width of 2 and a power of 75%. Cell debris was removed by centrifugation at 12000 rpm for 15 min, the supernatant was collected, aliquoted, and stored at -80°C. The energy buffer composition is shown in Table 1. After preparation, the buffer was aliquoted and stored at -80°C.

[0054] Table 1. Energy Buffer Components 3. Construction of Arsenic Ion Sensing Plasmids: ArsR (nucleotide sequence as shown in SEQ ID No. 2), ArsC (nucleotide sequence as shown in SEQ ID No. 3), and sensor plasmids using mScarlet red fluorescent protein (nucleotide sequence as shown in SEQ ID No. 8) as a reporter gene were constructed using PCR and Gibson Assembly methods. See plasmid maps below. Figure 7 .

[0055] 4. Preparation of linear DNA templates: Key expression modules of all plasmids were amplified by PCR. The purity of the bands was analyzed by agarose gel electrophoresis. Linear DNA templates were extracted using a DNA fragment recovery kit (TaKaRa MiniBEST DNA FragmentPurification Kit Ver.4.0), eluted with approximately 50 µL of pure water, with a concentration of approximately 200~400 ng / µL, aliquoted and stored at -20℃.

[0056] The primer pairs used for linearization of all plasmids were the same: forward primer VF2: 5'-TGCCACCTGACGTCTAAGAA-3', and reverse primer VR: 5'-ATTACCGCCTTTGAGTGAGC-3' (primers were ordered from Hangzhou Youkang Biotechnology Co., Ltd. and purified by PAGE).

[0057] In use, the DNA templates are divided into two groups: pre-expression and post-addition. The pre-expression group includes ArsR, ArsC, and LacZω (nucleotide sequences as shown in SEQ ID No. 7) (added during the colorimetric reaction); the post-addition DNA template includes: ParsRD -ecf11 (where P) arsRD The nucleotide sequence of [ef11] is shown in SEQ ID No. 1, and the nucleotide sequence of [ef11] is shown in SEQ ID No. 4. arsRD -mScarlet, P ecf11 -mScarlet(P ecf11 The nucleotide sequence is shown in SEQ ID No. 5), P arsRD -lacZα、P ecf11 -lacZα. The lacZα-containing line (nucleotide sequence shown in SEQ ID No. 6) is added only during the colorimetric reaction, while the line containing mScarlet is added only during fluorescence characterization. In the colorimetric reaction, the J101-lacZα template is added as a positive control to verify whether the environmental water sample significantly interferes with cell-free samples.

[0058] 5. Preparation of Cell-Free Sensing System: The cell-free system contains 30% (v / v) E. coli lysate, 25% (v / v) energy buffer, and the remaining volume consists of water, DNA templates, GamS, and CPRG (chromogenic substrate, added only during colorimetric sensor preparation). Unless otherwise specified, all DNA templates are at a concentration of 3 nM, GamS at 1 µM, and CPRG at 1.2 mg / mL.

[0059] First, calculate the DNA addition volume based on the pre-expressed and post-added DNA concentrations. Add the lysate, energy buffer, GamS, and pre-expressed DNA sequentially to the centrifuge tube, then add water to make up the remaining volume (subtracting the post-added DNA volume and CPRG volume (for colorimetric reaction only). The volume should be less than 5% of the standard cell-free volume to prevent significant interference with the cell-free system). Carefully mix by pipetting, avoiding air bubbles. Incubate the centrifuge tube in a 37°C metal bath or water bath for 30 minutes, then immediately place it on ice. Add the post-added DNA and CPRG colorimetric reaction substrate, and mix carefully. For different sensor combinations, the types and concentrations of DNA templates required are shown in Table 2. The fluorescence sensor will detect the DNA in the circuitry... lacZ⍺ Replace with mScarlet That's all.

[0060] Table 2. Sensor and DNA template concentrations (nM) Example 2: Fabrication and Packaging of Paper-Based Colorimetric Sensor 1. Paper-based sensor chip fabrication: A designed pattern was printed on Cytiva medium-speed filter paper (60×60 cm, 99-102-952, Cytiva) using a wax printer (ColorQube 8570, Xerox). Figure 8 (a) The wax-printed filter paper is sandwiched between two pieces of glass and heated on a 110°C constant-temperature soldering station for 10 seconds to melt the wax and evenly distribute it on both sides of the filter paper to form a hydrophobic barrier. Subsequently, these paper chips are sandwiched on a 3D-printed magnetic support, and 2 µL of 0.005% (mass fraction) BSA (bovine serum albumin) solution is added to each reaction zone, and the mixture is air-dried at 37°C (approximately 1 hour).

[0061] 2. Addition of cell-free sensing system: The pre-expressed cell-free colorimetric reaction system (containing CPRG and colorimetric reporter circuitry) from Example 1 was added. lacZ⍺ and lacZω The samples were rapidly added to the paper-based chip treated in step 1 in the following order: N (negative control), 1 (without amplifier circuit, weakest sensitivity), 2 (without amplifier circuit, moderate sensitivity), 3 (with amplifier circuit, highest sensitivity), and P (positive control). 2 µL was added to each reaction zone.

[0062] 3. Freeze-drying of sensor paper: Fixing the paper-based chip onto a 3D-printed magnetic bracket ( Figure 8 (b) Place the plastic culture dishes in an elevated position. Stack multiple plastic culture dishes containing sensor chips into an aluminum metal bucket (a metal bucket with an inner diameter of 100 mm, a thickness of 10 mm, and a height of 50 mm can hold four 90 mm plastic culture dishes), place metal blocks on top and bottom, and secure with tape. Store at -80°C. After about 1-2 hours, the freezer will be completely frozen, and both the metal blocks and the metal bucket will remain at a low temperature to prevent the internal sensing system from freezing and thawing, which could cause sensor failure. Place the entire device in a freeze dryer and freeze-dry overnight.

[0063] 4. Sensor packaging and storage: The freeze-dried paper-based chip and the 3D-printed magnetic bracket are pushed into a 3D-printed black light-proof hard shell, and vacuum-sealed together with silica gel desiccant and stored at 4℃.

[0064] Example 3: Detection and Semi-quantitative Result Reading Using a Paper-Based Colorimetric Sensor 1. Fix the paper-based sensor inside the 3D-printed magnetic holder, add 2 µL of sample to each well in sequence, place it in a transparent disposable plastic petri dish with a moistened paper towel at the bottom, wrap it with sealing film for two weeks to ensure that the inside is sealed and moisturized.

[0065] 2. Place the petri dish in a 30℃ incubator. After 2 hours, remove it and observe it with the naked eye or take a picture with your mobile phone and save the image.

[0066] 3. Visually interpret the current colorimetric results. If only the positive control shows color, it indicates that the arsenic ion concentration in the water sample is less than 10 ppb; if two spots show color, the arsenic ion concentration is 10-50 ppb; if three spots show color, the arsenic ion concentration is 50-100 ppb; if four spots show color, the arsenic ion concentration is greater than 100 ppb, indicating severe water pollution. If the negative control shows color, or the positive control does not show color, it indicates significant interference in the water sample, rendering the test invalid.

[0067] Deionized water and solutions containing arsenic ions of 10, 50, and 100 ppb were respectively added as samples to a paper-based arsenic ion sensor array using the above method. After 2 hours of incubation, the color pattern exhibited by the paper-based sensor array showed a clear "stepped response" pattern, consistent with the expected results. Simultaneously, the paper-based sensor array showed the same response level to both trivalent and pentavalent arsenic ions. Figure 6 (b) in the middle.

[0068] Using the method described above, aqueous solutions containing 50 ppb of arsenic, zinc, mercury, manganese, copper, lead, cadmium, iron, nickel, cobalt, chromium, and calcium ions were respectively added dropwise to paper-based arsenic ion sensors S2 and S3. After 2 hours of incubation, the paper-based sensor only produced a significant colorimetric response to the water sample containing arsenic ions, and no colorimetric response to other ions, demonstrating that the sensor has good arsenic ion specificity; at the same time, the amplifier circuit was not affected by interference from other metal ions, and the system has good robustness. Figure 6 (c in the text)

[0069] The paper-based sensor prepared according to the method in Example 2 (taking S2 as an example) was rehydrated with a 50 ppb arsenic ion solution after being stored at 4°C for 1, 4, 7, 14, 21, 30, and 60 days, respectively. After incubation for 2 hours, the paper-based sensor at all storage times showed normal color development and maintained an extremely low leakage level. This proves that the paper-based arsenic ion sensor can be stored at 4°C for more than 2 months without degrading its original performance. Figure 6 (d in the text)

[0070] The team collected four representative natural water samples contaminated with arsenic in Khurna, Bangladesh. The arsenic ion concentrations were measured to be 1.21 ppb, 16.0 ppb, 71.0 ppb, and 137.6 ppb, respectively. The colorimetric results of the paper-based sensor array in the field were as expected, with 1, 2, 3, and 4 spots producing colorimetric responses, respectively, demonstrating the feasibility of this sensor in practical detection applications. Figure 9 ).

[0071] The embodiments described above are merely specific examples of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention, such as selecting appropriate induction systems and output genes for different application scenarios. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A biosensor for arsenic ion synthesis based on a cell-free system, characterized in that, The module includes a cell-free arsenic ion sensing module, which is composed of the following functional units connected in sequence according to the signal transmission order: (a) A synthesis arsenic acid reduction module, used to directionally convert pentavalent arsenic into trivalent arsenic; (b) An arsenic ion sensing module comprising an arsenic-responsive promoter and an arsenic-responsive transcription factor, wherein the arsenic-responsive transcription factor undergoes a conformational change in the presence of trivalent arsenic, thereby relieving transcriptional repression of the arsenic-responsive promoter; (c) Signal gain processing module, containing Vibrio parahaemolyticus Vibrio parahaemolyticus extracellular functional σ factor ecf11 and its homologous promoter P ecf11 The output of the arsenic-responsive promoter is concatenated with the coding sequence of ecf11, wherein the P ecf11 Used to drive downstream gene expression; (d) A reporter module, containing a reporting element, for outputting a detectable signal; All of the above modules are integrated into a cell-free expression system.

2. The arsenic ion synthesis biosensor based on a cell-free system according to claim 1, characterized in that, The arsenic-responsive promoter is a P nucleotide sequence as shown in SEQ ID No.

1. arsRD ; The arsenic-responsive transcription factor is ArsR, with a nucleotide sequence as shown in SEQ ID No. 2; The synthetic arsenate reduction module contains an arsenate reductase ArsC with the nucleotide sequence shown in SEQ ID No. 3; The nucleotide sequence of the ecf11 is shown in SEQ ID No. 4, and the P ecf11 The nucleotide sequence is shown in SEQ ID No. 5; The reporter module comprises a complementary colorimetric system of LacZα and LacZω nucleotide sequences as shown in SEQ ID No. 6 and SEQ ID No. 7, or a fluorescent protein mScarlet as shown in SEQ ID No.

8. All genes used in the cell-free arsenic ion sensing module are linear DNA fragments, each fragment containing transcription terminator elements on both sides.

3. The arsenic ion synthesis biosensor based on a cell-free system according to claim 1, characterized in that, It also includes a semi-quantitative paper-based detection chip, which is formed by treating filter paper with a hydrophobic barrier to create five linearly arranged dot-matrix reaction zones, which are, in order, a negative control zone, a first arsenic ion sensing system zone, a second arsenic ion sensing system zone, a third arsenic ion sensing system zone, and a positive control zone; wherein the arsenic ion sensitivity of the first, second, and third arsenic ion sensing system zones increases sequentially, corresponding to arsenic ion concentrations of 100 ppb, 50 ppb, and 10 ppb, respectively. The color development mode of the chip is such that the number of reaction zones that change from yellow to purple is positively correlated with the concentration of arsenic ions in the water sample being tested.

4. The arsenic ion synthesis biosensor based on a cell-free system according to claim 1, characterized in that, The cell-free expression system was prepared using the BL21 Star (DE3) ΔlacZ strain, where BL21 Star (DE3) ΔlacZ was a knockout strain. lacZ The gene-bearing Escherichia coli BL21 Star (DE3); The lacZ The gene sequence is shown in SEQ ID No. 12 and is used for knockout. lacZ The gene sequence of the DNA fragment of the gene is shown in SEQ ID No.

13.

5. The arsenic ion synthesis biosensor based on a cell-free system according to claim 1, characterized in that, The constitutive promoters used to express LacZω, ArsR, and ArsC are selected from SJM901, J23101*, and J23101, respectively, and their nucleotide sequences are shown in SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 11, respectively. In the positive control, the J23101 promoter was used to express mScarlet or LacZα.

6. The arsenic ion synthesis biosensor based on a cell-free system according to claim 1, characterized in that, The cell-free arsenic ion sensing module was prepared using a pre-expression method. First, the DNA circuit containing the arsenic-responsive transcription factor ArsR was added to a cell-free system and pre-reacted at 37°C for 30 minutes; then, reporter element DNA and chromogenic substrate were added for colorimetric reaction. The ArsC and ArsR circuits in the synthetic arsenic acid reduction module are added simultaneously during the pre-expression stage to achieve simultaneous equivalent detection of pentavalent and trivalent arsenic.

7. The arsenic ion synthesis biosensor based on a cell-free system according to claim 1, characterized in that, By adjusting the concentration of each DNA template in the cell-free arsenic ion sensing module, the sensitivity and dynamic response range of the sensor to arsenic ions are changed, forming a sensing system with three different sensitivity gradients.

8. The application of the arsenic ion synthesis biosensor according to any one of claims 1-7 in the on-site detection of arsenic pollution in drinking water.

9. The application according to claim 8, characterized in that, When applying, the following steps are included: (1) Take the drinking water sample to be tested, which does not require pretreatment or only requires static sedimentation treatment; (2) Add the drinking water sample to be tested to each reaction zone of the semi-quantitative paper-based detection chip to rehydrate the freeze-dried cell-free arsenic ion sensing module. (3) Incubate the paper-based detection chip at 30°C for 2 hours; (4) Semi-quantitative interpretation can be made by observing the color changes of each reaction area with the naked eye, or by taking pictures with a smartphone for analysis.

10. A method for preparing an arsenic ion synthesis biosensor based on a cell-free system, characterized in that, include: (a) Construction of a cell-free arsenic ion sensing module: The linear DNA templates for synthesizing the arsenic acid reduction module, arsenic ion sensing module, signal gain processing module, and reporter module are mixed with a cell-free expression system, wherein the DNA templates contain the sequence elements shown in SEQ ID No. 1 to SEQ ID No. 11; (b) Preparation of semi-quantitative paper-based detection chip: A hydrophobic barrier was prepared on filter paper using paraffin printing technology to form five linearly arranged dot array reaction areas, which correspond to the negative control, the arsenic ion sensing system with three sensitivity gradients and the positive control, respectively. (c) Spot the cell-free sensing system obtained in step (a) onto the corresponding reaction area of ​​the chip in step (b), and freeze-dry it using the ice bucket low-temperature freeze-drying method to obtain a paper-based freeze-dried biosensor, namely the arsenic ion synthesis biosensor.

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