A portable thermostatic molecular diagnosis device based on high-efficiency photothermal conversion material for rapid field detection of SARS-CoV-2

Abstract

The outbreak of the novel coronavirus (SARS-CoV-2) has seriously harmed human health and economic development worldwide. Studies have shown that timely diagnosis and isolation are the most effective ways to prevent the spread of the epidemic. However, the current polymerase chain reaction (PCR) based molecular diagnostic platform has the problems of expensive equipment, high operation difficulty, and the need for stable power resources support, so it is difficult to popularize in low-resource areas. This study established a portable (<300 g), low-cost (<$10), and reusable molecular diagnostic device based on solar energy photothermal conversion strategy, which creatively introduces a sunflower-like light tracking system to improve light utilization, making the device suitable for both high and low-light areas. The experimental results show that the device can detect SARS-CoV-2 nucleic acid samples as low as 1 aM within 30 min.

Graphical abstract

1. Introduction

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has hit the world for three years, infecting more than 200 million people and killing millions more [[1], [2], [3]]. Studies have shown that timely diagnosis and isolation are the most effective ways to prevent the spread of SARS-CoV-2, and the low detection coverage in low-resource areas has been a major challenge for epidemic prevention and control [4,5]. The main reason is that the traditional polymerase chain reaction (PCR) requires expensive equipment ($ 60,000–150,000), a stable power supply (accurate thermal cycling), a professional operating environment, and skills (prevent aerosol pollution), so it is difficult to popularize in the field environment and low resource areas [6,7]. The recombinase polymerase amplification (RPA) and its derived recombinase aided amplification (RAA) are thermostatic nucleic acid amplification technologies that can efficiently amplify the template under the temperature of 37–44 °C [[8], [9], [10]]. The advantage of not requiring variable temperature cycling allows the RAA to be amplified very quickly, and the manufacturing cost of the accompanying equipment is greatly reduced, making it ideal for portable point-of-care (POC) molecular diagnostics [11,12].

The efficient amplification of RAA requires a constant reaction temperature of 37–42 °C, so the design of portable POCT devices is focused on providing stable energy in low-resource areas and field environments. Most portable molecular diagnostic devices currently rely on pre-prepared batteries or chemicals to generate heat [13,14]. This wasteful, one-off energy supply method does not guarantee a long-term energy supply in the field environment [15,16]. Given this problem, solar energy may be a better source for POC molecular diagnosis [[17], [18], [19], [20]]. In our previous work, we have successfully prepared various highly efficient photothermal conversion materials, which can rapidly increase the reaction temperature and maintain a constant temperature for a long time, and subsequent seawater desalination has verified this excellent performance [[21], [22], [23]]. From this, it can be inferred that the energy supply of photothermal materials will become a promising energy source for the sustainable utilization of molecular diagnostic equipment in the field.

Herein, a portable thermostatic molecular diagnostic device based on an efficient photothermal conversion material, which uses inexhaustible sunlight to provide energy, has been established in this study, which can effectively convert solar energy into thermal energy and maintain a constant temperature of 37–42 °C for RT-RAA reaction. The Experimental results show that the device can detect SARS-CoV-2 nucleic acid samples as low as 1 aM within 30 min. This device creative introduces the sunflower-like light tracking system, improving light utilization and making the device suitable for high and low light areas, so it is ideal for use in resource-poor areas and fieldwork environments.

2. Results and discussions

As shown in Scheme 1 , the RT-RAA amplification technology with constant low temperature (37–42 °C) can greatly reduce the requirements on the heating module, which is conducive to reducing the cost and complexity of the device, so it is more suitable for the portable molecular diagnostic device in low-resource areas and field environments. To fully use the sunlight, a sunflower-like automatic light tracker ensures that the photothermal reactor always faces the sun to generate enough heat to sustain the RT-RAA amplification reaction. The attached solar panels can store electricity in batteries that power the light-tracking system and an ultraviolet flashlight (observe the fluorescent results by the naked eye). This approach effectively reduces the need for generating efficiency compared to directly heating the RT-RAA reaction using electricity. The lightweight manufacturing materials are used to reduce the weight of the photothermal reactor to ensure that the photosensitive panels rotate smoothly with low energy consumption, so the entire device weighs only 300 g and is easy to carry around. In detail, the shell is made of foam, photothermal nanoparticles are grown in situ on melamine sponges, and the convex lens is made of lightweight plastic. In addition, the reverse transcription-RAA (RT-RAA) kit can be stored at room temperature for a long time after freeze-drying to remove moisture. With the help of the high photothermal performance of black gold sponge (BGS), the RT-RAA reaction can quickly reach the optimal reaction temperature of 36–44 °C within 5–10 min, and the whole RT-RAA detection time is only 30 min. Based on the above advantages, this portable molecular diagnostic device is suitable for rapidly detecting virus samples in low-resource areas and fieldwork environments.

Scheme 1.

The schematic of a portable thermostatic molecular diagnostic device for SARS-CoV-2 detection.

Photothermal conversion nanomaterials that could efficiently absorb sunlight are the key point of the photothermal RT-RAA amplification systems. In our study, the BGS with high photothermal conversion efficiency was prepared by in situ syntheses of gold nanoparticles on the melamine sponge substrate (95% of porosity). As shown in Fig. 1 a, the melamine sponge was placed into a mixture of 5% sodium citrate and 0.5% chloroauric acid aqueous, which could enter and fill the interspace of the melamine sponge by the capillary effect. Then, the sodium citrate reduces chloroauric acid to gradually form gold nanoparticles on the inner surface of the sponge over time. Fig. 1b shows the SEM images of BGS at different magnifications. The melamine sponge exhibits a three-dimensional multi-interspace structure, and the average size of the interspace is 30 μm, which provides favorable conditions for transporting sodium citrate and chloroauric acid reaction aqueous mixture. The SEM image with greater magnification shows that the size and morphology of the gold nanoparticles adsorbed on the interspace surface are irregular (nanocrystalline clusters, nanosheets, nanospheres, etc.), which is conducive to the efficient absorption of sunlight with different wavelengths by gold nanoparticles. Considering that the BGS is a black solid, its diffuse reflection absorption spectrum was obtained using a UV–Vis–NIR spectrophotometer with an integrating sphere attachment (UV-3600PLUS) (Fig. S1). As expected, the absorbance of the BGS was 93.6% in the range from 300 nm to 2500 nm and 93.7% in the range from 300 nm to 1400 nm. This result is consistent with most current studies. In addition, the BGS is stable enough to be reused, and subsequent stability tests have confirmed that the absorbance of BGS is not affected by extreme conditions such as strong acids and alkalis.

Fig. 1.

The BGS with high-efficiency photothermal conversion. (a) The manufacturing process of BGS by in situ synthesis. (b) The SEM images of BGS with different magnifications. (c) The temperature rises curve of the aqueous solution in the PCR tube wrapped with BGS under different light intensity (200, 400, 600, 800 W/m²) and (d) the lens used to raise the temperature of the reaction system under low light intensity (170–300 W/m²).

In the experiment to test the heating effect of the BGS on the RT-RAA reaction system, the PCR tube coated with the BGS sleeve was placed under different light intensities (shown in Fig. S2 Xenon lamp, solar-500, NBET; optical power density meter, CEL-NP2000, CEAULIGHT), and the temperature sensor (Digital thermometer, LCD 110, Hengshuichuangji) monitored the real-time temperature change of the aqueous solution in the PCR tube (Fig. S3). The results in Fig. 1c show that the BGS can rapidly increase the temperature of the aqueous solution from 25 °C to 36–42 °C (efficient RT-RAA amplification) within 5–10 min through photothermal conversion when the light intensity is > 400 W/m². However, the temperature of the aqueous solution is below 36 °C (inefficient RT-RAA amplification) when the light intensity is < 400 W/m². To further increase the temperature of the RT-RAA reaction at low light intensities (<400 W/m²), an additional convex lens (φ = 5 cm, f = 4 cm) to focus the weak light and increase the local light intensity, showing that the reaction temperature increased from 31-35 °C to 37–40 °C (Fig. 1d). Moreover, considering that the ambient temperature in practical application is not the ideal 25 °C, we have tested the temperature rise of the reaction system under different ambient temperatures. As shown in Fig. S4, when the ambient temperature is as low as 15 °C and 20 °C, the PCR tube wrapped with BGS can still rapidly warm up within 5–10 min and remain at about 37 °C, which is consistent with the temperature control effect of 25 °C, indicating that the fluctuation of external temperature will not lead to temperature control failure. This result is due to the stable photothermal conversion ability of the BGS, and the heat capacity of the air is low, so the ambient air temperature has little effect on the heating rate. Using these methods, we are expected to achieve efficient amplification of nucleic acid template by the RT-RAA reaction under different light intensities.

The SARS-CoV-2, a global infectious disease currently widely concerned, is the target template in this experiment, and the sequence information of the RAA primers and TwistAmp® Exo probe is shown in Table. S1. As shown in Fig. 2 a, gel electrophoresis images exhibited five comparative RT-RAA amplification products (10 fM of RNA template) to test the amplification efficiency of the RT-RAA under different conditions. For the high light (600 W/m²) experiment group, the clear 200 bp of the target band confirmed that the RT-RAA reaction efficiently finished nucleic acid amplification with the help of the BGS (lane 1). However, when the BGS was absent, the amplification efficiency decreased dramatically due to low temperature (lane 2). For the low light (200 W/m²) experiment group, the bright target band was obtained only when the BGS and convex lens were used together (lane 3), and the absence of either condition failed RT-RAA amplification due to the low temperature (lane 4 and lane 5). Since gel electrophoresis detection is unsuitable for field detection, we further designed the TwistAmp® Exo probe for fluorescence analysis. As shown in Fig. 2b, the RT-RAA reaction can rapidly detect SARS-CoV-2 standard sample as low as 1 aM within 20 min on the qPCR instrument, and there was a good linear relationship between the RNA template concentration and the fluorescence signal growth rate.

Fig. 2.

The nucleic acid detection on the portable thermostatic molecular diagnostic device. (a) The RT-RAA amplification is achieved using only the BGS under high light intensities (>400 W/m²); both the BGS and the lens are required for RT-RAA amplification in low light intensities (<400 W/m²). (b) The RT-RAA fluorescence detection method can realize the results read out by the end-point fluorescence intensity. (c) The photo of the portable thermostatic molecular diagnostic device. (d) The naked eye readout of molecular diagnostic results realized by ultraviolet flashlight.

Due to the photothermal conversion of the BGS requiring sufficient sunlight and the convex lens also needing to face the sun for good performance, the sunflower-like light tracking system was used as the auxiliary component of the portable molecular diagnostic device. As shown in Fig. 2c, the dual-axis light-tracking system of the portable molecular diagnostic device consists of a 51 single-chip, four photoresistors, a ULN2803, two-stepping motor, a LED lamp, five control buttons, a PCF 8591, a solar panel, a lithium battery, a charging module, an LCD 1602 screen, etc., and which was packaged and wrapped with the black PMMA plate. The attached Mov. 1 shows that as the position of the light source changes, the light-sensitive plate of the light tracking system constantly adjusts the angle to ensure that it is always facing the light source.

Since the load-bearing capacity of the plate is limited, we have chosen lightweight materials (sponges, plastic lenses, etc.) to make the photothermal converter. As shown in Fig. S5, the BGS was stuffed into a foam with a hole 2–3 cm deep in the middle, and a circular plastic convex lens (φ = 5 cm, f = 4 cm) was attached to the top surface of the foam to focus sunlight (The distance from the PCR tube to the lens is less than the focal length, and the focused light spot can completely cover the reaction zone, so the problem of uneven heating of the reaction zone will not occur). Before testing, the operator adds the 50 μL target RNA to the RT-RAA powder reagent and inserts the reaction tube into the interlayer of the BGS. After the detection equipment is activated, the photothermal converter and photosensitive plate will automatically turn to face the sun, and then the convex lens will focus the sunlight on the BGS, heating the RT-RAA reaction and activating the nucleic acid amplification. During the reaction, the fluorescence reporter sequences of TwistAmp®Exo can release free fluorescent molecules after being cleaved by exonuclease. After about 20 min of reaction, enough fluorescent molecules had accumulated that could be excited by an additional ultraviolet flashlight and then observed the detection result with the naked eye. As shown in Fig. 2d, the results are consistent with the fluorescence camera in the laboratory.

In field tests, rapid viral inactivation and RNA release must also be considered in addition to the miniaturization of molecular diagnostic devices [24]. Although the traditional magnetic bead and column nucleic acid extraction kits can obtain relatively pure templates from the virus, they are often difficult to operate, time-consuming, and require additional equipment (centrifuge, heater, etc.) [25]. In our experiments, we abandoned the RNA purification process and instead used the “direct amplification rapid nucleic acid extraction lysate (Hangzhou Zhongce Biotech)” to obtain the RNA template of the pseudovirus to reduce the difficulty of operation. As shown in Fig. S6, the magnetic bead extraction method and “direct amplification rapid nucleic acid extraction lysate” were both used to crack pseudovirus samples, and the little difference in Ct value between viral RNA obtained by these two methods for the qPCR test confirmed that the rapid lysate could be fully used for nucleic acid extraction in the field test.

To verify the validity and accuracy of the portable device, three simulated throat swab samples (samples 1#, 2#, and 3#) attached with different concentrations of pseudovirus were diagnosed in the field and laboratory using a portable molecular diagnostic device and PCR equipment, respectively. The field diagnosis process is shown in Fig. 3 a: a) The throat swab sample was directly placed into the rapid lysate and occasionally shaken for 10 min to release RNA; b) 5 μL of RNA suspension was mixed with 45 μL of buffer solution and then added into the PCR tube containing RT-RAA powder reagent (10% “direct amplification rapid nucleic acid extraction lysate” did not fail RT-RAA amplification); c) The portable device automatically points the sample at the sun and heat the RT-RAA reaction; d) Use the ultraviolet flashlight to excite and observe the detection results with the naked eye after 30 min of reaction. The pictures showed that sample 1# was negative, and samples 2# and 3# were positive. On the other hand, the diagnostic results were obtained by the standard processes of “magnetic bead extraction-PCR detection” after 2 h, which indicated that sample 1# was negative and the Ct values of sample 2# and 3# were 24 and 35, respectively (Fig. 3b). The comparison results confirm the accuracy and stability of portable detection equipment in field testing, and the detection time is shorter.

Fig. 3.

The comparison results between the portable devices for field and laboratory standard PCR tests. (a) The sample treatment process and naked eye fluorescence test results from the portable devices in the field test. (b) The detection result from the standard PCR method.

3. Conclusions

In summary, this study established a portable thermostable molecular diagnostic device based on gold nanoparticles with efficient photothermal conversion, which can directly heat the RT-RAA amplification reaction to 37–44 °C. Field tests show the device could detect virus samples as low as 1 aM within 30 min. Considering the current situation of insufficient energy supply in low-resource and field areas, we propose the following innovations to solve the problems in on-site detection: 1) The convex lens focuses the sunlight so that the instrument can also be used in low-light areas; 2) The sunflower-type light chasing system improves the utilization rate of sunlight and the stability of detection; 3) The machine weight is light (300 g), easy to carry for field detection; 4) The energy stored by the solar panel provides sufficient power support for the light-sensitive platform, which is capable of long-term detection tasks. 5) Depending on the endpoint fluorescence signal to read the results, the user can use an ultraviolet flashlight to make a visual judgment. Best of all, the device is powered by an inexhaustible supply of solar energy, so it is reusable. With no need for external stable power support and the low price of the whole machine (<$10), it is very suitable for use in resource-poor areas. The lightweight, portable testing equipment allows it to provide disease diagnosis services to temporary workers in the field or rapid on-site testing for food safety.

Credit author statement

Xinyao Yang: Methodology, Validation, Data curation, Manuscript preparation. Chuanghao Guo: Data curation, Manuscript preparation. Qianling Zhang: Project administration, Writing – review & editing. Yong Chen: Conceptualization, Methodology, Project administration, Funding acquisition, Writing – original draft, Supervision. Yizhen Liu: Conceptualization, Project administration, Funding acquisition, Writing – review & editing. Xueji Zhang: Project administration, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Qun Fang

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Data availability

Data will be made available on request.