Proof of Concept

1. Background

Currently, environmental pollution is a significant concern, with heavy metal contamination being a prominent issue in aquatic environments. It not only impacts aquatic ecosystems but also poses potential hazards to human drinking water sources. While various methods exist for heavy metal detection, most of them require specialized equipment and have stringent detection requirements. Moreover, effective pollution control methods are lacking, making it challenging to address and remediate pollution issues even when specific contaminated areas are identified. Some chemical-based methods can also lead to secondary pollution, complicating practical applications.

Cadmium contamination poses substantial environmental and health risks. Many countries have established mandatory emission standards to limit its impact on the environment. National standards such as "Hygienic Standard for Drinking Water" (GB/T5749-2006) and "Fishery Water Quality Standards" (GB11067-89) specify that the cadmium content should not exceed 0.005mg/L. Therefore, the analysis and measurement of cadmium in water are crucial.

Our project has developed a detection method based on heavy metal aptamers. By designing these nucleic acid aptamers in a specialized manner, we can initiate the expression of fluorescent protein markers and corresponding heavy metal-binding proteins using a neck-loop switch with aptamer nucleotide sequences. This approach allows for the precise detection of heavy metals and holds promise for pollution control, making it highly applicable.

2. Development of Paper-Based Sensors Using a Cell-Free Expression System

Detection of the presence and concentration of heavy metals through the expression of fluorescent proteins, while simultaneously expressing metal-specific binding proteins to adsorb heavy metals from the environment, aiming to reduce pollution and control contamination.

Construction and identification of the neck-loop switch plasmid

We have constructed neck-loop switch plasmids with a target sequence matching the cadmium aptamer nucleotide sequence. These neck-loop switch plasmids were identified using a restriction enzyme digestion method involving EcoR I and Hind III.

Figure 1.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.

These neck-loop switch plasmids are primarily used to express cadmium-binding proteins, specifically green fluorescent protein (GFP), in the BL21 strain.

The successful expression of the reporter proteins in BL21 indicates the interaction between these neck-loop switches and cadmium.

Figure 2. Expression of CBP and GFP protein in BL21 strain. A, Positive control of BL21 strain containing pET-22b-CBP-GFP plasmid. B, BL21 strain transfected with pET-22b-clip switch-CBP-GFP plasmid and Cd2+。

To determine the optimal detection concentration and related cultivation conditions, we conducted studies involving different cadmium concentrations and cultivation times.

Figure 3. Optimization comparison of different Cd2+ concentrations and cultivation times. A, Exploration of Cd detection limits; B, Exploration of the linear range of Cd detection

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 4. 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.

Research on Paper-Based Sensors Using a 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 5. 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.

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 6. 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 0.02 mg/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.