With the large amount of industrial sewage and human life wastewater discharge, heavy metal pollution of water has become a global environmental problem. Heavy metal pollution is not only a great threat to the ecological balance and the safety of drinking water, but also can act together with other pollutants in living organisms, presenting a complex joint effect in the organism, and ultimately causing comprehensive toxicity. Take our country, China, as an example, there has been no breakthrough in the treatment of cyanobacteria in Lake Taihu, which meanly because that the environmental sewage treatment is costly, and some pollution sources are difficult to detect quickly.

At present, heavy metal detection methods mainly include inductively coupled plasma emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectrometry (AAS), and atomic fluorescence spectrometry (AFS), etc. However, these detection methods are not convenient enough to carry out effective treatment in a short term, especially the basic cost is high, which is not conducive to the use of large-scale or high-frequency. We can also use biosensor technology to carry out simple, rapid, sensitive and accurate in situ dynamic monitoring and analysis of important pollutants in the environment, which has shown great advantages and potential. However, biosensors are too demanding to be used in practical applications. Synthetic biology has reawakened the potential of whole-cell microbial sensors.

Figure .1 Schematic diagram of the principle of Heavy Metal Cadmium's detection and adsorption

When there is a heavy metal cadmium in the environment, it can be combined with the ligand, the non-cell expression system in the paper base (filter paper), the expression of the cadmium binding protein and the green fluorescent protein, both the visual effect and the heavy metal cadmium in the environment, the double target of detection and purification.

1.Extraction of water environment DNA and amplification of target gene (cadmium binding protein)

Collect samples of water environment with potential pollution, use the corresponding DNA extraction kit, extract DNA, and gel electrophoresis detection, the results are shown below.

Figure.2 Gel electrophoresis profiles of DNA from aquatic environments

Figure.3 Results of cadmium binding protein CBP amplification

As shown in the above figure, primers were designed for PCR amplification according to the DNA sequence of cadmium-binding protein, and the corresponding target gene sequences were obtained for two of the samples, with a length of 1100bp.

Amplification and identification of cadmium-binding protein CBP gene fragment
The amino acid sequence corresponding to the binding protein gene is as follows.

MNIQIGELAKRTACPVVTIRFYEQEGLLPPPGRSRGNFRLYGEEHVERLQFIRHCRSLDMPLSDVRTLLSYRKRPDQDCGEVNMLLDEHIRQVESRIGALLELKHHLVELREACSGARPAQSCGILQGLSDCVCDTRGTTAHPSDGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKLE

According to its gene sequence, primers are designed to amplify from the total DNA.
The gene sequences used for cloning expression are as

follows.CATATGAACATCCAGATTGGCGAACTGGCAAAACGTACCGCATGTCCGGTTGTTACCATTCGTTTTTATGAACAGGAAGGTCTGCTGCCGCCGCCGGGTCGTAGCAGAGGTAATTTTCGTCTGTATGGTGAAGAACATGTTGAACGCCTGCAGTTTATTCGTCATTGTCGTAGTCTGGATATGCCGCTGAGCGATGTTCGCACCCTGCTGAGCTATCGTAAACGCCCGGATCAGGATTGTGGCGAAGTGAATATGCTGCTGGATGAACATATTCGCCAGGTTGAAAGTCGCATTGGTGCACTGCTGGAACTGAAACATCATCTGGTGGAACTGCGCGAAGCATGCAGCGGTGCCCGCCCTGCTCAGAGCTGCGGTATTCTGCAGGGCCTGAGTGATTGTGTTTGTGATACCCGTGGTACCACCGCACATCCGAGTGATGGTGGTGGCGGCAGTGGCGGCGGCGGTAGCATGGTTAGCAAAGGTGAAGAACTGTTTACCGGCGTTGTTCCGATTCTGGTGGAATTAGATGGCGATGTTAATGGTCATAAATTTTCTGTGAGTGGCGAAGGCGAAGGCGATGCCACCTATGGTAAACTGACCCTGAAATTCATTTGTACCACCGGCAAACTGCCGGTTCCGTGGCCGACCCTGGTGACCACCCTGACCTATGGCGTTCAGTGTTTTAGCCGTTATCCGGATCACATGAAACAGCATGATTTCTTTAAAAGTGCAATGCCGGAAGGTTATGTGCAGGAACGTACCATTTTCTTTAAAGATGATGGCAATTACAAGACCCGTGCCGAAGTGAAATTTGAAGGCGATACCCTGGTGAATCGTATTGAACTGAAAGGTATTGATTTCAAAGAGGATGGTAATATTCTGGGTCATAAACTGGAATATAACTATAATAGCCACAACGTTTACATCATGGCAGATAAACAGAAAAATGGTATTAAGGTTAACTTCAAGATCCGCCATAATATTGAAGATGGCAGTGTTCAGCTGGCCGATCATTATCAGCAGAATACCCCGATTGGCGATGGCCCGGTGCTGCTGCCGGATAATCATTATCTGAGTACCCAGAGCGCACTGAGCAAAGATCCGAATGAAAAACGTGATCACATGGTGCTGCTGGAATTTGTGACCGCAGCAGGCATTACCCTGGGTATGGATGAACTGTATAAACTCGAG

2.Construction of cyclic expression plasmid

Firstly, HindIII and EcoRI double digestion was performed, then CBP was ligated with it, and the ligand transformed E.coli BL21(DE3) receptor cells, and screened with ampicillin to obtain a certain number of transformants. The plasmid was verified by enzymatic digestion. As shown in the figure below, the size of the enzymatically digested target gene fragment was as expected.

Figure .4 Recombinant plasmid zymography 1, Double zymography of target gene; 2, Plasmid.

The expression vector map used in this project

3.Expression and detection of target proteins

For the successful realisation of the cell-free expression system, we first carried out the validation of traditional cellular expression.

Figure .5 Expression and validation of cadmium binding protein

A: Growth of transformed colonies; B: SDS-PAGE to detect the fusion protein; C: Western Blot to analyse the expression of fusion protein.

The above results show that the cadmium binding protein obtained better expression after transforming E. coli and can be used for subsequent experimental research.

4.Construction and validation of neckloop switch plasmid

In order to carry out the visual detection of heavy metal cadmium, we used GFP fluorescent protein as a reporter gene, showing yellow-green colour, and we constructed pET-22b-GFP as a positive control.

The constructed pET-22b-GFP was digested with EcoRI and HindIII restriction endonucleases, and PCR was performed using the flanking sequences of GFP.The gene fragment of GFP was 780bp, which was detected using agarose gel electrophoresis, and the results showed the same length as expected (shown below).

Figure .6 Recognition of pET-22b-neck-loop switch-CBP-GFP. M: Marker; 1: pET-22b-neck-loop

switch-CBP-GFP plasmid with EcoRI and HindIII restriction enzyme digestion, 2: pET-22b neck-loop switch-CBP-GFP plasmid.

The BL21 strain transfected with the plasmid was cultured on a petri dish, and the results showed normal expression of fluorescent proteins, and these bacteria were used as a positive control for the transfection of the neckloop switch plasmid. The expression results are shown below:

Figure 7 .Expression of CBP and GFP protein in BL21 strain. 1: Positive control of BL21 strain containing pET-22b-CBP-GFP plasmid. 2: BL21 strain transfected with pET-22b-clip switch-CBP-GFP plasmid and Cd

The neckloop switch plasmid was designed and constructed based on the sequence of GFP, the sequence of cadmium-binding protein and the sequence of cadmium aptamer, which is capable of producing a yellow-green reporter protein. The plasmid was synthesised by EcoRI-HindIII cloning site and cloned into pET-22b expression vector to detect cadmium.

This neckloop switch plasmid was validated by restriction endonuclease digestion experiments. The insertion sequence was 780bp and this result was verified by agarose gel electrophoresis.

5.Induction of neckloop plasmid with chromium ions to express fluorescent reporter gene in BL21 strain

The pET-22b-neckloop switch-CBP-GFP plasmid was transfected into BL21 strain (below). In the absence of cadmium ion induction, the expression of the GFP gene was not triggered in the BL21 strain, thus failing to express the GFP protein or the amount of GFP protein expression, indicating that the neckloop switch was effective. Subsequently, cadmium ions were transfected into BL21 strains containing the pET-22b-neckloop

switch-CBP-GFP plasmid. It was found that some clones successfully transfected with cadmium ions showed yellow-green colour.

Figure .8 GFP expression in BL21 strains. A: strain without transfection of cadmium ion; B: BL21 strain transfected with both cadmium ion and switch plasmid.

6.Exploration of the sensitivity of cadmium ion detection in the expression system

After transfecting the neck-ring switch plasmid in BL21 strain, we explored the culture conditions of the strain and optimised the concentration of cadmium ions and the incubation time.

Since the reporter gene contains colour, we can optimize the reaction conditions by colour. Considering the detection sensitivity and the later paper-based detection system, it is crucial to find the lowest chromium ion detection concentration, and we mainly explored the detection concentration. The corresponding experimental results are shown below.

Figure 9 Optimization comparison of different Cd2+ concentrations and cultivation times. A, Exploration of Cd detection limits; B, Exploration of the linear range of Cd detection.

From the above graph, it can be seen that with the increase of Cd2+ concentration, the expression of fluorescent protein became more and more obvious, and there was a good linear correlation between the concentration of 0.02mg/L and 1mg/L.

7.Exploration of reaction time of cell-free expression system
After identifying the optimal detection concentration and related cultivation conditions, we utilized ultrasonic treatment to obtain cell extract. This extract, mixed with ATP, phosphoenolpyruvate (PEP), amino acids, and other components, was used for inducing the expression of target proteins in the presence of cadmium ions. We investigated protein expression at various reaction times and found that 1 hour was the most suitable reaction time.

Figure 10 Exploration of the expression time of fluorescent proteins in the cell-free expression system.
A: Expression of paper-based fluorescent proteins; B: Analysis of fluorescence intensity at different times

8.Research on paper-based detection sensor based on cell-free expression system
Filter paper is an easily accessible, cost-effective, and convenient biological analysis material. After soaking filter paper in bovine serum albumin and drying it, we applied a cell-free reaction system to the filter paper. The paper was then freeze-dried to create paper-based sensors, which can be stored for an extended period. During testing, we added cadmium ion solutions to filter paper sensors containing neck-loop switch plasmids and incubated them at 37°C for 1 hour, allowing the proteins to be expressed.

Figure 11 Expression of the reporter gene GFP induced by different cadmium ion concentrations
The results demonstrate that the expression of the reporter gene GFP can be activated with a cadmium solution of 0.02 mg/L, making it suitable for rapid detection of cadmium contamination in typical aquatic environments.

9.Testing of Real Water Pollution Samples
To further validate the practical usability of our prepared sensors, we collected some polluted water samples from the environment. We conducted tests using paper-based sensors and observed faint expression of fluorescent proteins (as shown in the figure below), indicating the potential applicability of these sensors for broader use.

Figure .12 Detection using paper-based sensors in real water pollution samples

In Summary:

In this study, we have established a paper-based biosensor for the detection of cadmium in aquatic environments using cadmium aptamers. Our results demonstrate that we can detect cadmium solutions with concentrations as low as 0.02mg/L using these paper-based sensors, which exhibit sensitivity comparable to traditional methods for analyzing polluted water. This approach is simple and convenient and holds significant potential for the detection and potential remediation of heavy metal pollution in aquatic environments.

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