Emerging biosensing and transducing techniques for potential applications in point-of-care diagnostics

Abstract

With the deepening of our understanding in life science, molecular biology, nanotechnology, optics, electrochemistry and other areas, an increasing number of biosensor design strategies have emerged in recent years, capable of providing potential practical applications for point-of-care (POC) diagnosis in various human diseases. Compared to conventional biosensors, the latest POC biosensor research aims at improving sensor precision, cost-effectiveness and time-consumption, as well as the development of versatile detection strategies to achieve multiplexed analyte detection in a single device and enable rapid diagnosis and high-throughput screening. In this review, various intriguing strategies in the recognition and transduction of POC (from 2018 to 2021) are described in light of recent advances in CRISPR technology, electrochemical biosensing, and optical- or spectra-based biosensing. From the perspective of promoting emerging bioanalytical tools into practical POC detecting and diagnostic applications, we have summarized key advances made in this field in recent years and presented our own perspectives on future POC development and challenges.

Fig. 1. The components of a POC device including a potential target, probe, sensing tool and transducer, signal acquisition and processing, and their corresponding elements. Biological targets mainly consist of nucleic acids, whole cells, bacteria/virus particles, proteins and small molecules, while probes usually contain synthetic or natural antibodies, oligonucleotides, proteins and other supra/small molecules. Sensing tools primarily refer to the sensing materials and platforms such as nanomaterials, hydrogels and CRISPR. Transducers convert the probe recognition into signals, which are then displayed on readable devices.

Fig. 2. The potential POC sensing platform for nucleic acids enabled by CRISPR-Cas9. (A) Schematic reaction mechanism of simultaneous dual-gene amplifying of SARS-CoV-2 by RT-RPA and sensing by LFA. Reproduced from ref. with permission. Copyright 2021 Wiley-VCH. Using RT-RPA, the E gene and Orf1ab gene regions of extracted samples are amplified and labeled with biotin and digoxin, respectively. In a typical test, the aforementioned complexes are dropped onto the sample pad and allowed to flow to the conjugate pad, where the AuNP–DNA probes hybridize to the scaffold sequence. Finally, due to the accumulation of AuNPs, the colorful bands could be observed with the naked-eye. (B) Overview of a multiplexable LEOPARD platform for parallel RNA detection. (i) Design process of Rptr; (ii) NcrRNA generation process; (iii) scheme of the multiplexed detection process for RNAs of interest. Rptrs convert sensed RNAs into ncrRNAs, which would direct Cas9 to match DNA. Cas9 binding or cleavage of a DNA sequence then indicate the presence of the sensed RNA in the sample. Reproduced from ref. with permission. Copyright 2021 American Association for the Advancement of Science.

Fig. 3. CRISPR-Cas12/Cas13 based biosensing strategies used in nucleic acid detection. (A) Schematic illustration of typical collateral cleavage-based CRISPR-Cas systems, SHERLOCK and DETECTR. The Cas protein's trans-cleavage activity is activated when the crRNA binds to the nucleic acid target, resulting in the cleavage of multiple reporter molecules to achieve detection. (B) Schematic diagram of the DETECTR workflow for SARS-CoV-2. Conventional RNA extraction was used as an input to DETECTR, which was visualized by a fluorescent reader or lateral flow strip. This assay employs RT-LAMP to perform simultaneous reverse transcription and isothermal amplification of RNA extracted from swabs in universal transport medium (UTM), followed by Cas12 detection of predefined coronavirus sequences and cleavage of a reporter molecule to confirm virus detection. Reproduced from ref. with permission. Copyright 2020 Springer Nature. (C) Amplification-free quantitative detection method for combining crRNAs targeting multiple regions of the viral RNA enhances sensitivity towards SARS-CoV-2 with a portable, mobile phone-based device. By combining multiple crRNAs to increase Cas13a activation, this assay can analyze the change in fluorescence over time with a mobile phone camera in a compact device that includes low-cost laser illumination and collection optics rather than solely endpoint fluorescence. Ref. with permission. Copyright 2021 Elsevier Inc. (D) Design of a CRISPR-based wearable device. Distributed continuous sensing of garment activity can be achieved through multi-bundle imaging. The various optical fibers carrying the output emission signals from different sensors can be routed into a single bundle for centralized imaging analysis. Ref. with permission. Copyright 2021 Springer Nature.

Fig. 4. CRISPR-based strategies used in the detection of non-nucleic acid targets. (A) Molecular translation mechanisms for the CRISPR-based POC detection for non-nucleic acid targets. (B) Detection strategy and workflow of the fDNAs-regulated CRISPR-Cas12a sensor for adenosine triphosphate (ATP) using a hand-hold portable device. Target binding to the fDNA in the presence of ATP can induce the DNA activator to dehybridize from the fDNA and become an “open activator” that binds to and activates the Cas12a. Reproduced from ref. with permission. Copyright 2020 American Chemical Society. (C) Scheme of CLISA for femtomolar high throughput detection of proteins assisted by CRISPR-Cas13a. The capture antibody binds to the antigen of interest first, followed by a biotinylated detection antibody that is streptavidin-linked to a biotin-dsDNA template. Following transcription, the amplified RNAs activate trans-cleavage, allowing for detection. Reproduced from ref. with permission. Copyright 2020 American Chemical Society.

Fig. 5. Electrochemical-based POC strategies used in nucleic acid detection. (A) Scheme illustration of the synthetic gene detection method that combines cell-free transcription and translation systems with engineered gene circuits on nanostructured microelectrodes. In short, this approach employs toehold switch-based RNA sensors which will express one of ten restriction-enzyme-based reporters in the presence of trigger RNA. Afterwards, restriction enzymes cleave annealed reporter DNA which was followed by releasing reporter DNA labelled with the redox reporter (blue circle). After the capture of reporter DNA by probe DNA on nanostructured microelectrodes, it will generate an electrochemical signal. Reproduced from ref. with permission. Copyright 2020 Springer Nature. (B) Schematic representation of steps involved in miRNA detection by using Au@mNPs. Au@MNPs are modified with methylene-blue-labelled probe DNA which is complementary to target miRNA. Unhybridized sequences are washed away after magnetic separation of Au@MNPs from the solution. Magnetically collected Au@MNPs (both hybridized and unhybridized) on the surface of a gold microelectrode provide the electrochemical signal. Reproduced from ref. with permission. Copyright 2018 Springer Nature.

Fig. 6. Electrochemical-based POC biosensors for the detection of proteins and bacteria. (A) Reagentless electrochemical biosensor of protein detection by monitoring the kinetics of transport of a molecular pendulum complex constructed with dsDNA, a specific antibody and a redox reporter. This approach relies on the motion of an inverted molecular pendulum that exhibits field-induced transport. The motion is regulated by the presence of bound analytes which is quantified by the sensor's electric field-mediated transport. Reproduced from ref. with permission. Copyright 2021 Springer Nature. (B) Workflow of an immuno-affinity amperometric method for the detection of bacterial infection which is composed of three steps: (1) Bacterial capture and enrichment from complex matrices; (2) chemical reduction and (3) amperometric read-out. Reproduced from ref. with permission. Copyright 2018 Wiley-VCH.

Fig. 7. (A) Overall fabrication and workflow of competition-based universal photonic crystal hydrogel biosensors by using antibody–antigen interactions. The photonic crystal hydrogel sensors are modified with the complex of antibody–antigen which both were covalently linked on the scaffold hydrogel. After the detection of free targets, the breakage between immobilized antibody and antigen will cause significant expansion and a color change of the hydrogel from the contracted state which can be detected by the naked eye and smart phone. This universal design enabled the successful quantitative detection of small molecules, peptides and proteins with the naked eye and smart phone. Reproduced from ref. with permission. Copyright 2020 American Chemical Society. (B) Rapid POC microengineered device technology for TBI biodiagnostics. The left figure shows the construction of this device. The middle figure shows the optical image and SEM image of the copper master used for fabrication of the PDMS, and a photograph of the microfluidic device with an input of 1–2 drops of whole human blood. Briefly, the whole blood will be separated by an optofluidic lab-on-a-chip to plasma. The optical information enhanced by the microfabricated SERS chip is sent to a PC to display the collected spectra using a designed portable spectrometer. Reproduced from ref. with permission. Copyright 2020 Springer Nature. (C) AuNPs/ZnONRs/C chip for SERS analysis of amniotic fluids to detect prenatal diseases. The procedures are as follows: collection of amniotic fluids; dropping 2 μL of amniotic fluid onto the AuNPs/ZnONRs/C chip and real-time Raman measurement. Reproduced from ref. with permission. Copyright 2018 American Chemical Society.

Fig. 8. (A) Schematic illustration of the plasmonic-fluorophore structure and its working principle as a biolabel to enhance the FL and S/N ratio of immunoassays. The plasmonic-fluorophore consists of a plasmonically-active core (AuNR), a polymer shell as a spacer layer, light emitters, and biotin served as a universal biorecognition element. In the detection process, the analytes are recognized by the detection antibodies on the microarray and in the sample mixture to generate a sandwich structure, followed by binding with streptavidin and plasmonic fluorophores to achieve detection with enhanced optical signals. Reproduced from ref. with permission. Copyright 2020 Springer Nature. (B) Schematic depiction of the LSPR-based biosensor for label-free detection of tear biomarkers based on different affinity interactions. Based on the sensing strategy that the dependence of the refractive index changes on the amount of protein bound is critical, little to no shift in LSPR wavelength is expected for low affinity contacts or low protein concentrations. Larger red shifts in the LSPR wavelength are expected as protein concentration or affinity increases. Reproduced from ref. with permission. Copyright 2018 American Chemical Society.

Fig. 9. (A) Schematic illustration of dSimoa. From left to right shown in the scheme, the main structure was composed of antibody-coated paramagnetic beads, the target protein, the biotinylated detector antibody, the labeled streptavidin–DNA conjugate, DNA polymerase and the probe. RCA is conducted on the beads after the development of a single immunocomplex on antibody–magnetic beads–streptavidin–DNA sandwiches to construct a lengthy concatemer connected to each immunocomplex. The concentrated beads are then dropcast onto a microscope slide and fluorescence imaging of the dropcast film is used to count single target molecules. Reproduced from ref. with permission. Copyright 2020 American Chemical Society. (B) Schematic showing the design of the paper-based ratiometric fluorescence analytical device and the chemical structure of 4MC. In this assay, the concentration of HSA was determined by comparing the ratios of emission from probes in aggregated and monomeric states, which results in a visible red-to-green color change on a simple, portable paper-based analytical device developed by integrating the identification probe into the detecting pad. Reproduced from ref. with permission. Copyright 2020 Wiley-VCH. (C) Illustration of the broad adaptability of SENSR and its easy reconfiguration and rapid development for various pathogens. The assay relies on a sustained isothermal reaction cascade producing an RNA aptamer that binds to a fluorogenic dye. T7 RNA polymerase transcribes the RNA aptamer from a promoter DNA probe and a reporter DNA probe that hybridize with the target single-stranded RNA sequence via the SplintR ligase. After the one-pot isothermal reaction, the complex shows strong fluorescence in the reaction tube. Two pathogenic microbes and three viruses were targeted by redesigning the probe sequences. Reproduced from ref. with permission. Copyright 2020 Springer Nature.

Fig. 10. (A) The design principle of a BRET sensor for POC testing of NADPH and the results of parallel analysis of 96 phenylalanine samples using naked eye. For this method, the metabolite is oxidized by nicotinamide adenine dinucleotide phosphate, and the sensor changes color when the reduced cofactor is present, allowing for metabolite measurement using a digital camera. Reproduced from ref. with permission. Copyright 2018 American Association for the Advancement of Science. (B) Ratiometric fluorescent lateral flow immunoassay (RFLFIA) strip under daylight (i), UV light (ii), and SEM images (iii) after treatment with negative and positive assays for acute myocardial infarction (50 ng mL⁻¹). In this design, the RFLFIA strip works according to the ratiometric change of the fluorescence signal, arising from blending of fluorescence emitted by two composite nanostructures conjugated to capture and probe antibodies and the inner filter effect of gold nanoparticles. Finally, detection is achieved by using a custom smartphone-based analytical device. Reproduced from ref. with permission. Copyright 2021 Wiley-VCH.