Ultrasensitive and amplification-free detection of SARS-CoV-2 RNA using an electrochemical biosensor powered by CRISPR/Cas13a.

This study proposed a CRISPR/Cas13a-powered electrochemical multiplexed biosensor for detecting SARS-CoV-2 RNA strands. Current SARS-CoV-2 diagnostic methods, such as reverse transcription PCR (RT-PCR), are primarily based on nucleic acid amplification (NAA) and reverse transcription (RT) processes, which have been linked to significant issues such as cross-contamination and long turnaround times. Using a CRISPR/Cas13a system integrated onto an electrochemical biosensor, we present a multiplexed and NAA-free strategy for detecting SARS-CoV-2 RNA fragments. SARS-CoV-2 S and Orf1ab genes were detected in both synthetic and clinical samples. The CRISPR/Cas13a-powered biosensor achieved low detection limits of 2.5 and 4.5 ag/µL for the S and Orf1ab genes, respectively, successfully meeting the sensitivity requirement. Furthermore, the biosensor's specificity, simplicity, and universality may position it as a potential rival to RT-PCR.

Rapid, multiplexed, and nucleic acid amplification-free detection of SARS-CoV-2 RNA using an electrochemical biosensor.

Considering the worldwide health crisis associated with highly contagious severe respiratory disease of COVID-19 outbreak, the development of multiplexed, simple and rapid diagnostic platforms to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is in high demand. Here, a nucleic acid amplification-free electrochemical biosensor based on four-way junction (4-WJ) hybridization is presented for the detection of SARS-CoV-2. To form a 4-WJ structure, a Universal DNA-Hairpin (UDH) probe is hybridized with two adaptor strands and a SARS-CoV-2 RNA target. One of the adaptor strands is functionalized with a redox mediator that can be detected using an electrochemical biosensor. The biosensor could simultaneously detect 5.0 and 6.8 ag/µL of S and Orf1ab genes, respectively, within 1 h. The biosensor was evaluated with 21 clinical samples (16 positive and 5 negative). The results revealed a satisfactory agreement with qRT-PCR. In conclusion, this biosensor has the potential to be used as an on-site, real-time diagnostic test for COVID-19.

A MoS2@Ti3C2Tx MXene hybrid-based electrochemical aptasensor (MEA) for sensitive and rapid detection of Thyroxine.

In the present study, a MoS2@Ti3C2Tx MXene hybrid-based electrochemical aptasensor (MEA) was introduced for sensitive and rapid quantification of Thyroxine (T4). T4 is a crucial hormone and plays a key role in various body functions. Therefore, there is high demand for an accurate, sensitive, and rapid method for the detection of T4. To construct the aptasensor, a nano-hybrid (NH) consisting of Ti3C2Tx MXene and MoS2 nanosheets (NS) was synthesized, and applied to a carbon electrode surface, followed by the electroplating of gold nanostructures (GN). The smart combination of Ti3C2Tx MXene and MoS2NS enhanced the physiochemical properties of the electrode surface, as well as provided a building block to form 3D GN. The 3D architecture of the GN offered a unique substrate to capture numerous T4 aptamer molecules, which consequently amplified the signal by nearly 6-fold. The MEA quantified thyroxine with a limit of detection (LOD) of 0.39 pg/mL over a dynamic range ((7.8 × 10-1) to (7.8 × 106)) pg/mL within 10 min. Moreover, the MEA successfully detected T4 in human serum samples. Lastly, the results obtained from the aptasensor were compared with those from the ELISA standard method. The comparative analysis showed good agreement between the two methods.

Recent advances in sensitive surface-enhanced Raman scattering-based lateral flow assay platforms for point-of-care diagnostics of infectious diseases.

This review reports the recent advances in surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) platforms for the diagnosis of infectious diseases. As observed through the recent infection outbreaks of COVID-19 worldwide, a timely diagnosis of the disease is critical for preventing the spread of a disease and to ensure epidemic preparedness. In this regard, an innovative point-of-care diagnostic method is essential. Recently, SERS-based assay platforms have received increasing attention in medical communities owing to their high sensitivity and multiplex detection capability. In contrast, LFAs provide a user-friendly and easily accessible sensing platform. Thus, the combination of LFAs with a SERS detection system provides a new diagnostic modality for accurate and rapid diagnoses of infectious diseases. In this context, we briefly discuss the recent application of LFA platforms for the POC diagnosis of SARS-CoV-2. Thereafter, we focus on the recent advances in SERS-based LFA platforms for the early diagnosis of infectious diseases and their applicability for the rapid diagnosis of SARS-CoV-2. Finally, the key issues that need to be addressed to accelerate the clinical translation of SERS-based LFA platforms from the research laboratory to the bedside are discussed.

Detachable microfluidic device implemented with electrochemical aptasensor (DeMEA) for sequential analysis of cancerous exosomes.

The quantification of cancer-derived exosomes has a strong potential for minimally invasive diagnosis of cancer during its initial stage. As cancerous exosomes form a small fraction of all the exosomes present in blood, ultra-sensitive detection is a prerequisite for the development of exosome-based cancer diagnostics. Herein, a detachable microfluidic device implemented with an electrochemical aptasensor (DeMEA) is introduced for highly sensitive and in-situ quantification of cancerous exosomes. To fabricate the aptasensor, a nanocomposite was applied on the electrode surface followed by electroplating of gold nanostructures. Subsequently, an aptamer against an epithelial cell adhesion molecule is immobilized on the electrode surface to specifically detect cancer-specific exosomes. A microfluidic vortexer is then constructed and implemented in the sensing system to increase the collision between the exosomes and sensing surface using hydrodynamically generated transverse flow. The microfluidic vortexer was integrated with the aptasensor via a 3D printed magnetic housing. The detachable clamping of the two different devices provides an opportunity to subsequently harvest the exosomes for downstream analysis. The DeMEA has high sensitivity and specificity with an ultra-low limit of detection of 17 exosomes/µL over a wide dynamic range (1 × 102 to 1 × 109) exosomes/µL in a short period. As proof of the concept, the aptasensor can be separated from the 3D printed housing to harvest and analyze the exosomes by real-time polymerase chain reaction. Moreover, the DeMEA quantifies the exosomes from plasma samples of patients with breast cancer at different stages of the disease. The DeMEA provides a bright horizon for the application of microfluidic integrated biosensors for the early detection of cancerous biomarkers.

All-in-one platform for salivary cotinine detection integrated with a microfluidic channel and an electrochemical biosensor.

Medical disorders caused by second-hand smoke are a major public health concern worldwide. To estimate the level of second-hand smoke exposure, salivary diagnostics for cotinine analysis is a compelling alternative in conventional diagnostics using bio-fluids, such as blood and urine, owing to its simple and non-invasive collection method. However, there are several critical issues, such as tedious multisteps, demand for expertise, and field unavailability to collect and transport the purified saliva for further analysis. Here, an all-in-one platform is presented to simply collect real human saliva and directly deliver it onto the biosensing surface. The platform consists of a commercial cotton-swab-type collector, 3D-printed housing, and microfluidic channel integrated with an electrochemical competitive immunosensor to evaluate the level of salivary cotinine. The immunosensor is based on a competitive binding assay between cotinine-conjugated horseradish peroxidase (C-HRP) and cotinine for anti-cotinine binding sites. The current responses obtained from the HRP-thionine-H2O2 system decreased proportionally to the cotinine concentration. This immunosensor successfully detected its target over a range of 1 × 10-1 to 1 × 104 pg ml-1 with a low limit of detection of 6 × 10-2 pg ml-1 and a limit of quantification of 1 × 10-1 pg ml-1. In addition, the platform is applicable to various commercial cotton-swab-type saliva collectors and can successfully transfer the saliva in wide flow rates ranging from 0.1 to 30 ml min-1 without leakage or damage to the sensing surface. Furthermore, the practicality of the proposed platform was evaluated by measuring cotinine in real human saliva from eight non-smokers. The concentration of cotinine was from 45.7 to 890.8 pg ml-1, which was in good agreement with that measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The introduced all-in-one platform represented a reliable performance delivering simple and practical steps in salivary diagnostics.

Progress in Circulating Tumor Cell Research Using Microfluidic Devices.

Circulating tumor cells (CTCs) are a popular topic in cancer research because they can be obtained by liquid biopsy, a minimally invasive procedure with more sample accessibility than tissue biopsy, to monitor a patient's condition. Over the past decades, CTC research has covered a wide variety of topics such as enumeration, profiling, and correlation between CTC number and patient overall survival. It is important to isolate and enrich CTCs before performing CTC analysis because CTCs in the blood stream are very rare (0⁻10 CTCs/mL of blood). Among the various approaches to separating CTCs, here, we review the research trends in the isolation and analysis of CTCs using microfluidics. Microfluidics provides many attractive advantages for CTC studies such as continuous sample processing to reduce target cell loss and easy integration of various functions into a chip, making "do-everything-on-a-chip" possible. However, tumor cells obtained from different sites within a tumor exhibit heterogenetic features. Thus, heterogeneous CTC profiling should be conducted at a single-cell level after isolation to guide the optimal therapeutic path. We describe the studies on single-CTC analysis based on microfluidic devices. Additionally, as a critical concern in CTC studies, we explain the use of CTCs in cancer research, despite their rarity and heterogeneity, compared with other currently emerging circulating biomarkers, including exosomes and cell-free DNA (cfDNA). Finally, the commercialization of products for CTC separation and analysis is discussed.

A microfluidic electrochemical aptasensor for enrichment and detection of bisphenol A.

Bisphenol A (BPA) is an organic monomer used to make common consumer goods such as plastic containers, sports equipment, and cosmetics which are heavily produced worldwide. A growing interest has been drawn to general public as BPA is one of the major endocrine disrupting chemicals threating human health. To date, numerous BPA sensors have been attempted to be developed but important challenges still remained such as limited linearity range, easy to use, and long term response time. To address the present issues, a microfluidic channel should be integrated into an electrochemical aptasensor and it is called Geometrically Activated Surface Interaction (GASI) chip. The vigorous generation of the micro-vortex in the GASI fluidic chamber provides the high collision chances between BPA and anti-BPA aptamer (BPAPT) and consequently more BPA molecules can be captured on the aptasensor surface, which finally results in high sensitivity of the aptasensor. To construct the integrated aptasensor, a miniaturized gold electrode is fabricated using shadow mask and e-beam evaporation process. Afterward, BPAPT is immobilized on a nanostructured gold electrode via thiol chemistry, and other terminus of the aptamer is labeled with a ferrocene (Fc) redox probe. Then, the microfluidic channel is mounted over the miniaturized gold electrode to introduce and enrich BPA to the aptasensor. Upon the specific interaction between BPA and its aptamer, configuration of aptamer is changed so that Fc tag approaches to the electrode surface and direct oxidation signal of Fc and BPA are followed as analytical signals. The unique microfluidic integrated electrochemical aptasensor delivers a wide linear dynamic range over 5 × 10-12 to 1 × 10-9 M, with a limit of detection 2 × 10-13 M. This aptasensor provides a precise platform for simple, selective and more importantly rapid detection of BPA. Such kind of sensing platforms can serve as a fertile ground for designing miniaturized portable sensors.

Ultrasensitive detection of human liver hepatocellular carcinoma cells using a label-free aptasensor.

Liver cancer is one of the most common cancers in the world and has no effective cure, especially in later stages. The development of a tangible protocol for early diagnosis of this disease remains a major challenge. In the present manuscript, an aptamer-based, label-free electrochemical biosensor for the sensitive detection of HepG2, a hepatocellular carcinoma cell line, is described. The target cells are captured in a sandwich architecture using TLS11a aptamer covalently attached to a gold surface and a secondary TLS11a aptamer. The application of TLS11a aptamer as a recognition layer resulted in a sensor with high affinity for HepG2 cancer cells in comparison with control cancer cells of human prostate, breast, and colon tumors. The aptasensor delivered a wide linear dynamic range over 1 × 10(2) to 1 × 10(6) cells/mL, with a detection limit of 2 cells/mL. This protocol provides a precise method for sensitive detection of liver cancer with significant advantages in terms of simplicity, low cost, and stability.

Aptamer-based electrochemical biosensor for detection of adenosine triphosphate using a nanoporous gold platform.

In spite of the promising applications of aptamers in the bioassays, the development of aptamer-based electrochemical biosensors with the improved limit of detection has remained a great challenge. A strategy for the amplification of signal, based on application of nanostructures as platforms for the construction of an electrochemical adenosine triphosphate (ATP) aptasensor, is introduced in the present manuscript. A sandwich assay is designed by immobilizing a fragment of aptamer on a nanoporous gold electrode (NPGE) and its association to second fragment in the presence of ATP. Consequently, 3, 4-diaminobenzoic acid (DABA), as a molecular reporter, is covalently attached to the amine-label of the second fragment, and the direct oxidation signal of DABA is followed as the analytical signal. The sensor can detect the concentrations of ATP as low as submicromolar scales. Furthermore, 3.2% decrease in signal is observed by keeping the aptasensor at 4 °C for a week in buffer solution, implying a desirable stability. Moreover, analog nucleotides, including GTP, UTP and CTP, do not show serious interferences and this sensor easily detects its target in deproteinized human blood plasma.

Aptamer-conjugated silver nanoparticles for electrochemical detection of adenosine triphosphate.

The capability of silver nanoparticles (SNP) as redox tag in the construction of an electrochemical aptasensor for the detection of adenosine triphosphate (ATP) is investigated in the present manuscript. To construct the aptasensor, a well-known ATP binding aptamer (ABA) splits into two segments. The first amino-labeled segment of the aptamer was covalently immobilized on 3-mercaptopropionic acid modified gold electrode surface by the formation of carbodiimide bond. The second segment was modified by SNPs and associated with the first segment in the presence of ATP. The direct oxidation signal of SNPs is followed as the analytical signal to detect ATP. The sandwich assay shows a suitable signal gain and importantly, a good response time. The sensor can detect the concentrations of ATP as low as micromolar scales with a desirable stability under optimum conditions. Furthermore, analog nucleotides including GTP, UTP and CTP, do not show serious interferences and this sensor readily detects its target in a complex media such as human blood plasma.

Design and construction of a label free aptasensor for electrochemical detection of sodium diclofenac.

The present manuscript describes a label free electrochemical aptasensor for the detection of sodium diclofenac (DCF). In order to construct the biosensor, the amino-functionalized diclofenac binding aptamer (DBA) was covalently immobilized on the surface of the glassy carbon electrode (GCE). The conformation of the DBAs on the surface of the electrode is changed when this is exposed to different concentrations of DCF. The introduction of DCF induces an alteration in the conformation of the surface immobilized DBA and causes a decrease in the charge transfer resistance of the aptasensor. However, the charge transfer resistance is increased by incubation of GCE/DBA/DCF in the secondary DBA. The changes in the charge transfer resistance have been monitored using the voltammetric and electrochemical impedance spectroscopic (EIS) techniques. The aptasensor shows two different linear dynamic ranges over 0-5.0 µM and 10 µM to 1mM, and the sensitivity of 15.7 kΩ µM(-1) and detection limit of 2.7 × 10(-7)M were obtained. The validity of the method and applicability of the aptasensor were successfully evaluated by detection of DCF in a blood serum sample without interference from the sample matrix. Furthermore, the aptasensor has shown good stability.