Molecular Origins of Long-Term Changes in a Competitive Continuous Biosensor with Single-Molecule Resolution.

Biosensing by particle motion is a biosensing technology that relies on single-molecule interactions and enables the continuous monitoring of analytes from picomolar to micromolar concentration levels. However, during sensor operation, the signals are observed to change gradually. Here, we present a comprehensive methodology to elucidate the molecular origins of long-term changes in a particle motion sensor, focusing on a competitive sensor design under conditions without flow. Experiments were performed wherein only the particles or only the surfaces were aged in order to clarify how each individual component changes over time. Furthermore, distributions of particle motion patterns and switching activity were studied to reveal how particle populations change over timespans of several days. For a cortisol sensor with anticortisol antibodies on the particles and cortisol analogues on the sensing surface, the leading hypotheses for the long-term changes are (i) that the particles lose antibodies and develop nonspecific interactions and (ii) that analogue molecules dissociate from the sensing surface. The developed methodologies and the acquired insights pave a way for realizing sensors that can operate over long timespans.

Real-time continuous monitoring of dynamic concentration profiles studied with biosensing by particle motion.

Real-time monitoring-and-control of biological systems requires lab-on-a-chip sensors that are able to accurately measure concentration-time profiles with a well-defined time delay and accuracy using only small amounts of sampled fluid. Here, we study real-time continuous monitoring of dynamic concentration profiles in a microfluidic measurement chamber. Step functions and sinusoidal oscillations of concentrations were generated using two pumps and a herringbone mixer. Concentrations in the bulk of the measurement chamber were quantified using a solution with a dye and light absorbance measurements. Concentrations near the surface were measured using a reversible cortisol sensor based on particle motion. The experiments show how the total time delay of the real-time sensor has contributions from advection, diffusion, reaction kinetics at the surface and signal processing. The total time delay of the studied real-time cortisol sensor was ∼90 seconds for measuring 63% of the concentration change. Monitoring of sinusoidal cortisol concentration-time profiles showed that the sensor has a low-pass frequency response with a cutoff frequency of ∼4 mHz and a lag time of ∼60 seconds. The described experimental methodology paves the way for the development of monitoring-and-control in lab-on-a-chip systems and for further engineering of the analytical characteristics of real-time continuous biosensors.

High-Throughput Single-Molecule Sensors: How Can the Signals Be Analyzed in Real Time for Achieving Real-Time Continuous Biosensing?

Single-molecule sensors collect statistics of single-molecule interactions, and the resulting data can be used to determine concentrations of analyte molecules. The assays are generally end-point assays and are not designed for continuous biosensing. For continuous biosensing, a single-molecule sensor needs to be reversible, and the signals should be analyzed in real time in order to continuously report output signals, with a well-controlled time delay and measurement precision. Here, we describe a signal processing architecture for real-time continuous biosensing based on high-throughput single-molecule sensors. The key aspect of the architecture is the parallel computation of multiple measurement blocks that enables continuous measurements over an endless time span. Continuous biosensing is demonstrated for a single-molecule sensor with 10,000 individual particles that are tracked as a function of time. The continuous analysis includes particle identification, particle tracking, drift correction, and detection of the discrete timepoints where individual particles switch between bound and unbound states, yielding state transition statistics that relate to the analyte concentration in solution. The continuous real-time sensing and computation were studied for a reversible cortisol competitive immunosensor, showing how the precision and time delay of cortisol monitoring are controlled by the number of analyzed particles and the size of the measurement blocks. Finally, we discuss how the presented signal processing architecture can be applied to various single-molecule measurement methods, allowing these to be developed into continuous biosensors.

Electrochemical genosensor for the specific detection of SARS-CoV-2.

Infection caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for the Coronavirus disease (COVID-19) and the current pandemic. Its mortality rate increases, demonstrating the imperative need for acute and rapid diagnostic tools as an alternative to current serological tests and molecular techniques. Features of electrochemical genosensor devices make them amenable for fast and accurate testing closer to the patient. This work reports on a specific electrochemical genosensor for SARS-CoV-2 detection and discrimination against homologous respiratory viruses. The electrochemical biosensor was assembled by immobilizing thiolated capture probes on top of maleimide-coated magnetic particles, followed by specific target hybridization between the capture and biotinylated signaling probes in a sandwich-type manner. The probes were rigorously designed bioinformatically and tested in vitro. Enzymatic complexes based on streptavidin-horseradish peroxidase linked the biotinylated signaling probe to render the biosensor electrochemical response. The genosensor showed to reach a sensitivity of 174.4 µA fM-1 and a limit of detection of 807 fM when using streptavidin poly-HRP20 enzymatic complex, detected SARS-CoV-2 specifically and discriminated it against homologous viruses in spiked samples and samples from SARS-CoV-2 cell cultures, a step forward to detect SARS-CoV-2 closer to the patient as a promising way for diagnosis and surveillance of COVID-19.

Gold nanoparticle/DNA-based nanobioconjugate for electrochemical detection of Zika virus.

The development of a stable nanobioconjugate based on gold nanoparticles (AuNPs) linked to single-strand DNA (ssDNA) is reported for amplification of the electrochemical signal of a Zika virus (ZIKV) genetic material-based bioassay, with high sensitivity. The genosensor was assembled either at a screen-printed gold electrode (SPAuE) or a screen-printed carbon electrode decorated with hierarchical gold nanostructures (SPCE/Au), with Ru3+ as an electrochemical reporter. The genosensor response, interrogated by differential pulse voltammetry (DPV) at the transient current density, was linear from 10 to 600 fM and from 500 fM to 10 pM of the target, with a sensitivity of 2.7 and 2.9 µA cm-2 M-1 and a limit of detection of 0.2 and 33 fM at the SPAuE and SPCE/Au, respectively. The resultant genosensor detected ZIKV genetic material in raw serum samples from infected patients, with no sample pretreatment in a polymerase chain reaction amplification-free assay. The proposed ultrasensitive nanobioconjugate-based system offers a step forward to the diagnosis of the ZIKV, closer to the patient, and holds the potential for signal amplification in biosensing of a myriad of applications.Graphical abstract.

Nanobioconjugates for Signal Amplification in Electrochemical Biosensing.

Nanobioconjugates are hybrid materials that result from the coalescence of biomolecules and nanomaterials. They have emerged as a strategy to amplify the signal response in the biosensor field with the potential to enhance the sensitivity and detection limits of analytical assays. This critical review collects a myriad of strategies for the development of nanobioconjugates based on the conjugation of proteins, antibodies, carbohydrates, and DNA/RNA with noble metals, quantum dots, carbon- and magnetic-based nanomaterials, polymers, and complexes. It first discusses nanobioconjugates assembly and characterization to focus on the strategies to amplify a biorecognition event in biosensing, including molecular-, enzymatic-, and electroactive complex-based approaches. It provides some examples, current challenges, and future perspectives of nanobioconjugates for the amplification of signals in electrochemical biosensing.

Genosensors for differential detection of Zika virus.

Zika virus (ZIKV) is considered an emerging infectious disease of high clinical and epidemiological relevance. The epidemiological emergency generated by the virus in Latin America and Southeast Asia in 2014 evidenced the urgent need for rapid and acute diagnostic tools. The current laboratory diagnosis of ZIKV is based on molecular and serological methods. However, molecular tools need expensive and sophisticated equipment and trained personnel; and serological detection may suffer from cross-reactivity. In this context, genosensors offer an attractive alternative for field-ready, early and accurate diagnosis of ZIKV. This work reports on the development of genosensors for the differential detection of ZIKV and its discrimination from dengue (DENV) and chikungunya (CHIKV) homologous arboviruses. We designed specific capture and signal probes by bioinformatics, and prove their specificity to amplify the target genetic material by the polymerase chain reaction (PCR). The designed biotinylated capture and digoxigenin (Dig)-labeled signal probes hybridized the target in a sandwich-type format. An anti-Dig antibody labeled with the horseradish peroxidase (HRP) enzyme allowed for both optical and electrochemical detection. The genosensors detected the ZIKV genetic material in spiked serum, urine, and saliva samples and cDNA from infected patients, discriminating them from the DENV and ZIKV genetic material. The proposed system offers a step forward to the differential diagnosis of the ZIKV, closer to the patient, very promising for diagnosis and surveillance of this rapidly emerging disease.