Continuous Biosensing Research Articles

Continuous Biosensor Based on Particle Motion: How Does the Concentration Measurement Precision Depend on Time Scale?

Continuous biosensors measure concentration-time profiles of biomolecular substances in order to allow for comparisons of measurement data over long periods of time. To make meaningful comparisons of time-dependent data, it is essential to understand how measurement imprecision depends on the time interval between two evaluation points, as the applicable imprecision determines the significance of measured concentration differences. Here, we define a set of measurement imprecisions that relate to different sources of variation and different time scales, ranging from minutes to weeks, and study these using statistical analyses of measurement data. The methodology is exemplified for Biosensing by Particle Motion (BPM), a continuous, affinity-based sensing technology with single-particle and single-molecule resolution. The studied BPM sensor measures specific small molecules (glycoalkaloids) in an industrial food matrix (potato fruit juice). Measurements were performed over several months at two different locations, on nearly 50 sensor cartridges with in total more than 1000 fluid injections. Statistical analyses of the measured signals and concentrations show that the relative residuals are normally distributed, allowing extraction and comparisons of the proposed imprecision parameters. The results indicate that sensor noise is the most important source of variation followed by sample pretreatment. Variations caused by fluidic transport, changes of the sensor during use (drift), and variations due to different sensor cartridges and cartridge replacements appear to be small. The imprecision due to sensor noise is recorded over few-minute time scales and is attributed to stochastic fluctuations of the single-molecule measurement principle, false-positive signals in the signal processing, and nonspecific interactions. The developed methodology elucidates both time-dependent and time-independent factors in the measurement imprecision, providing essential knowledge for interpreting concentration-time profiles as well as for further development of continuous biosensing technologies.

Long-term and Continuous Plasmonic Oligonucleotide Monitoring Enabled by Regeneration Approach.

The demand for continuous monitoring of biochemical markers for diagnostic purposes is increasing as it overcomes the limitations of traditional intermittent measurements. This study introduces a method for long-term, continuous plasmonic biosensing of oligonucleotides with high temporal resolution. Our method is based on a regeneration-based reversibility approach that ensures rapid reversibility in less than 1 minute, allowing the sensor to fully reset after each measurement. We investigated label-free and AuNP enhancements for different dynamic ranges and sensitivities, achieving a limit of detection down to pM levels. We developed a regeneration-based reversibility approach for continuous biosensing, optimizing buffer conditions using the Taguchi method to achieve rapid, consistent reversibility, ensuring reliable performance for long-term monitoring. We detected oligonucleotides in buffered and complex solutions, including undiluted and unfiltered human serum, for up to 100 sampling cycles in a day. Moreover, we showed the long-term stability of the sensor for monitoring capabilities in buffered solutions and human serum, with minimal signal value drift and excellent sensor reversibility for up to 9 days. Our method opens the door to new prospects in continuous biosensing by providing insights beyond intermittent measurements for numerous analytical and diagnostic applications.

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.

Integrated Signal Amplification on a Fiber Optic SPR Sensor Using Duplexed Aptamers.

Throughout the past decades, fiber optic surface plasmon resonance (FO-SPR)-based biosensors have proven to be powerful tools for both the characterization of biomolecular interactions and target detection. However, as FO-SPR signals are generally related to the mass that binds to the sensor surface, multistep processes and external reagents are often required to obtain significant signals for low molecular weight targets. This increases the time, cost, and complexity of the respective bioassays and hinders continuous measurements. To overcome these requirements, in this work, cis-duplexed aptamers (DAs) were implemented on FO-SPR sensors, which underwent a conformational change upon target binding. This induced a spatial redistribution of gold nanoparticles (AuNPs) upon specific target binding and resulted in an amplified and concentration-dependent signal. Importantly, the AuNPs were covalently conjugated to the sensor, so the principle does not rely on multistep processes or external reagents. To implement this concept, first, the thickness of the gold fiber coating was adapted to match the resonance conditions of the surface plasmons present on the FO-SPR sensors with those on the AuNPs. As a result, the signal obtained due to the spatial redistribution of the AuNPs was amplified by a factor of 3 compared to the most commonly used thickness. Subsequently, the cis-DAs were successfully implemented on the FO-SPR sensors, and it was demonstrated that the DA-based FO-SPR sensors could specifically and quantitatively detect an ssDNA target with a detection limit of 230 nM. Furthermore, the redistribution of the AuNPs was proven to be reversible, which is an important prerequisite for continuous measurements. Altogether, the established DA-based FO-SPR bioassay holds much promise for the detection of low molecular weight targets in the future and opens up possibilities for FO-SPR-based continuous biosensing.

A Wireless, Regeneratable Cocaine Sensing Scheme Enabled by Allosteric Regulation of pH Sensitive Aptamers.

A key challenge for achieving continuous biosensing with existing technologies is the poor reusability of the biorecognition interface due to the difficulty in the dissociation of analytes from the bioreceptors upon surface saturation. In this work, we introduce a regeneratable biosensing scheme enabled by allosteric regulation of a re-engineered pH sensitive anti-cocaine aptamer. The aptamer can regain its affinity with target analytes due to proton-promoted duplex-to-triplex transition in DNA configuration followed by the release of adsorbed analytes. A Pd/PdHx electrode placed next to the sensor can enable the pH regulation of the local chemical environment via electrochemical reactions. Demonstration of a "flower-shaped", stretchable, and inductively coupled electronic system with sensing and energy harvesting capabilities provides a promising route to designing wireless devices in biointegrated forms. These advances have the potential for future development of electronic sensing platforms with on-chip regeneration capability for continuous, quantitative, and real-time monitoring of chemical and biological markers.

Paving the way towards continuous biosensing by implementing affinity-based nanoswitches on state-dependent readout platforms.

Continuous biosensors provide real-time information about biochemical processes occurring in the environment of interest and are therefore highly desirable in research, diagnostics and industrial settings. Although remarkable progress has been made in the field of biosensing, most biosensors still rely on batch processes and, thus, are not suited to perform continuous measurements. Recently, however, it has been shown that by combining affinity-based nanoswitches with state-dependent readout platforms, the necessity for batch processes can be overcome and affinity-based continuous biosensing can be achieved. In this review, we first provide an overview of affinity-based continuous biosensing and discuss the required components to achieve this goal. More specifically, we summarize the strategies that have been applied to develop and tune both protein and nucleic acid-based switches, as well as readout strategies that can be applied in combination with the former. Afterwards, biosensors in which both elements were already integrated and hence enabled continuous measurements are reviewed. We also discuss the challenges and opportunities associated with each approach and therefore believe this review can help to encourage and guide future research towards continuous biosensing.

Unrestrained and Continuous Biosensing for Medical and Health Sciences

Unrestrained and Continuous Biosensing for Medical and Health Sciences

Next-Generation Continuous Metabolite Sensing toward Emerging Sensor Needs.

This article discusses the emergent biosensor technology focused on continuous biosensing of metabolites by non-invasive sampling of body fluids emphasized on physiological monitoring in mobility-constrained populations, resource-challenged settings, and harsh environments. The boom of innovative ideas and endless opportunities in healthcare technologies has transformed traditional medicine into a sustainable link between medical practitioners and patients to provide solutions for faster disease diagnosis. The future of healthcare is focused on empowering users to manage their own health. The confluence of big data and predictive analysis and the internet of things (IoT) technology have shown the potential of converting the abundant health profile data amassed from medical diagnosis of patients into useable information, whilst allowing caregivers to provide suitable treatment plans. The implementation of the IoT technology has opened up advanced approaches in real-time, continuous, remote monitoring of patients. Wearable, point-of-care biosensors are the future roadmap to providing direct, real-time information of health status to the user and medical professionals in this digitized era.

Wireless battery-free wearable sweat sensor powered by human motion.

Wireless wearable sweat biosensors have gained huge traction due to their potential for noninvasive health monitoring. As high energy consumption is a crucial challenge in this field, efficient energy harvesting from human motion represents an attractive approach to sustainably power future wearables. Despite intensive research activities, most wearable energy harvesters suffer from complex fabrication procedures, poor robustness, and low power density, making them unsuitable for continuous biosensing. Here, we propose a highly robust, mass-producible, and battery-free wearable platform that efficiently extracts power from body motion through a flexible printed circuit board (FPCB)-based freestanding triboelectric nanogenerator (FTENG). The judiciously engineered FTENG displays a high power output of ~416 mW m-2 Through seamless system integration and efficient power management, we demonstrate a battery-free triboelectrically driven system that is able to power multiplexed sweat biosensors and wirelessly transmit data to the user interfaces through Bluetooth during on-body human trials.

Multiplexed Continuous Biosensing by Single-Molecule Encoded Nanoswitches.

Single-molecule techniques have become impactful in bioanalytical sciences, though the advantages for continuous biosensing are yet to be discovered. Here we present a multiplexed, continuous biosensing method, enabled by an analyte-sensitive, single-molecular nanoswitch with a particle as a reporter. The nanoswitch opens and closes under the influence of single target molecules. This reversible switching yields binary transitions between two highly reproducible states, enabling reliable quantification of the single-molecule kinetics. The multiplexing functionality is encoded per particle via the dissociation characteristics of the nanoswitch, while the target concentration is revealed by the association characteristics. We demonstrate by experiments and simulations the multiplexed, continuous monitoring of oligonucleotide targets, at picomolar concentrations in buffer and in filtered human blood plasma.

An electrochemical aptamer-based sensor prepared by utilizing the strong interaction between a DNA aptamer and diamond.

Stable and continuous biosensing of electroactive species in vivo has been achieved by using boron-doped diamond (BDD) electrodes owing to their outstanding electrochemical properties. However, the present problem in biosensing using BDD electrodes is how to specifically measure/detect the target molecules, including electrochemically inactive species. A possible solution is to fabricate an electrochemical aptamer-based (E-AB) sensor using a BDD electrode. In a preliminary investigation, we found that DNA aptamers strongly adsorb on the BDD surface and the aptamer-adsorbed BDD apparently worked as an E-AB sensor. The present study reports the performance of the aptamer-adsorbed BDD electrode as an E-AB sensor. Doxorubicin (DOX), a widely used chemotherapeutic, was chosen as a target molecule. The sensor could be prepared by just dipping BDD in an aptamer solution for only 30 min, and the electrochemical signals were dependent on the DOX concentration. The adsorption of DNA was strong enough for continuous measurements and even a sonication treatment. Such behaviors were not observed when using gold and glassy carbon electrodes. In a kinetic measurement, distortion by a sluggish response was observed for both association and dissociation phases, indicating that the interaction between DOX and the aptamer involves several kinetic processes. By fitting to a Langmuir isotherm, a limit of detection of 49 nM and a maximum detectable concentration of 2.3 μM were obtained. Although the sensitivity was lower than those of the well-established E-AB sensors of gold, the values are within a drug's therapeutic range. Overall, the present work demonstrates that a DNA aptamer and a BDD electrode is an effective combination for an E-AB sensor with stable sensitivity, and a wide variety of DNA aptamers can be applied without any special treatment.

Continuous cytometric bead processing within a microfluidic device for bead based sensing platforms

A microfluidic device for continuous biosensing based on analyte binding with cytometric beads is introduced. The operating principle of the continuous biosensing is based on a novel concept named the "particle cross over" mechanism in microfluidic channels. By carefully designing the microfluidic network the beads are able to "cross-over" from a carrier fluid stream into a recipient fluid stream without mixing of the two streams and analyte dilution. After crossing over into the recipient stream, bead processing such as analyte-bead binding may occur. The microfluidic device is composed of a bead solution inlet, an analyte solution inlet, two washing solution inlets, and a fluorescence detection window. To achieve continuous particle cross over in microfluidic channels, each microfluidic channel is precisely designed to allow the particle cross over to occur by conducting a series of studies including an analogous electrical circuit study to find optimal fluidic resistances, an analytical determination of device dimensions, and a numerical simulation to verify microflow structures within the microfluidic channels. The functionality of the device was experimentally demonstrated using a commercially available fluorescent biotinylated fluorescein isothiocyanate (FITC) dye and streptavidin coated 8 microm-diameter beads. After, demonstrating particle cross over and biotin-streptavidin binding, the fluorescence intensity of the 8 microm-diameter beads was measured at the detection window and linearly depends on the concentration of the analyte (biotinylated FITC) at the inlet. The detection limit of the device was a concentration of 50 ng ml(-1) of biotinylated FITC.

Biosensors in Wine Production Monitoring

ABSTRACT An overview of probe-type and flow-through biosensors, together with biosensing systems, both batch and continuous biosensing systems, for monitoring typical species in wine processes is presented with the aim of showing the advantages and disadvantages involved in the use of each type of devices. Thus, biosensors and biosensing systems for the determination of ethanol (individual or together with that of other compounds), organic acids, glycerol, reducing sugars, acetaldehyde and sulfur dioxide/sulfite anions are reviewed and critically compared. The versatility and capability of continuous biosensing systems as compared with biosensors, particularly with probe-type biosensors working in a discontinuous, batch manner, is demonstrated.