Detection of heavy metal contamination and remediation of the corresponding environments remain major challenges today. In this study, we present a novel and portable filter paper-based biosensor that can directly detect heavy metals such as cadmium in polluted water. The sensor consists of a stem-loop structure containing a cadmium aptamer nucleic acid and a fluorescent protein expression and regulatory gene. The DNA sequence of the stem-loop structure serves as a switch for fluorescent protein expression, and when cadmium is present, the fluorescent protein is expressed, and the amount of expression can react to a certain extent to the amount of pollutants, and the whole detection and protein expression system is on a strip of filter paper, which allows rapid detection and adsorption and purification of polluted water. Lay the foundation for further pollutant treatment.

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.1 Gel electrophoresis profiles of DNA from aquatic environments.

Figure.2 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 1100 bp.

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

1.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.4 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.

Construction and validation of neck loop 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.5 Construction and validation of linear plasmids by PCR and restriction enzyme digestion. a, double digestion neck loop switch plasmid; b, pET-22b(+)-neck loop switch-CBP-GFP plasmid; M, molecular weight Marker

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.6 Expression of fluorescent protein in BL21 bacteria transfected with pET-22b(+)-CBP-GFP plasmid.

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

Induction of neck loop plasmid with chromium ions to express fluorescent reporter gene in BL21 strain

The pET-22b(+)-neck loop 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 neck loop 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.7 GFP expression in BL21 strains.A: strain without transfection of cadmium ion; B: BL21 strain transfected with both cadmium ion and switch plasmid.

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.8 A, Effect of chromium ion concentration on fluorescent protein expression in BL21 strain transfected with neck loop switch plasmid, transfection concentrations from left to right are: 0.01mg/L, 0.02mg/L, 0.05mg/L, 0.1mg/L, 0.5mg/L, 1mg/L; B, linear correlation plots.

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 0.02 mg/L and 1mg/L.

Exploration of reaction time of cell-free expression system

After clarifying the optimal detection concentration and related culture conditions, the cell extracts were treated with ultrasonic waves to obtain the extracts, which were mixed with ATP, PEP, amino acids, etc., and cadmium ions were added for the induction of the expression of the target proteins, and the expression under different reaction times was explored. The results showed that there was basically no significant change in the fluorescence signal after 1 hour, so 1 hour was determined as the most suitable reaction time.

Figure.9 Exploration of fluorescent protein expression time in cell-free expression system.
A, Expression of paper-based fluorescent protein; B, Analysis of fluorescence intensity at different times.

Research on paper-based detection sensor based on cell-free expression system
Filter paper is a bioanalytical material that is easily accessible, inexpensive, and convenient for transport and storage. The filter paper is soaked in bovine serum albumin, dried, and then the cell-free reaction system is added dropwise to the filter paper, freeze-dried to form a paper-based sensor, which can be stored for a long time. For detection, an aqueous solution containing cadmium ions is added dropwise to the filter paper sensor containing the neck loop switch plasmid, and the protein is expressed by reacting at 37°C for 1 hour.

Figure.10 Expression of reporter gene GFP induced by different concentrations of cadmium ions.
The results show that the expression of reporter gene GFP can be activated with 0.02mg/L cadmium aqueous solution, which can be used for rapid detection of cadmium pollution in routine water environment.

Detection of actual water environment pollution samples
In order to further verify the practical usability of our prepared sensor, we collected some contaminated water samples from the environment and detected them using the paper-based sensor, which showed that the expression of some fluorescent proteins could be seen vaguely (shown in the following figure). It illustrates the possibility of the sensor's extended application.

Figure.11 Paper-based sensor detection in real water environment pollution samples.

In Summary

We established a paper-based biosensor in this study to detect cadmium contamination in aqueous environments through cadmium aptamers. The results showed that we were able to detect cadmium in aqueous solutions as low as 0.02 mg/L using the paper-based sensor, which is comparable to the sensitivity of conventional methods for detecting contaminated water, and can satisfy the detection of routinely contaminated water.
This method is simple and convenient, and is of great significance and potential promotion value for the pollution detection and potential treatment of heavy metals in the water environment.