Real-time detection of mAb aggregates in an integrated downstream process.

The implementation of continuous processing in the biopharmaceutical industry is hindered by the scarcity of process analytical technologies (PAT). To monitor and control a continuous process, PAT tools will be crucial to measure real-time product quality attributes such as protein aggregation. Miniaturizing these analytical techniques can increase measurement speed and enable faster decision-making. A fluorescent dye (FD)-based miniaturized sensor has previously been developed: a zigzag microchannel which mixes two streams under 30 s. Bis-ANS and CCVJ, two established FDs, were employed in this micromixer to detect aggregation of the biopharmaceutical monoclonal antibody (mAb). Both FDs were able to robustly detect aggregation levels starting at 2.5%. However, the real-time measurement provided by the microfluidic sensor still needs to be implemented and assessed in an integrated continuous downstream process. In this work, the micromixer is implemented in a lab-scale integrated system for the purification of mAbs, established in an ÄKTA™ unit. A viral inactivation and two polishing steps were reproduced, sending a sample of the product pool after each phase directly to the microfluidic sensor for aggregate detection. An additional UV sensor was connected after the micromixer and an increase in its signal would indicate that aggregates were present in the sample. The at-line miniaturized PAT tool provides a fast aggregation measurement, under 10 min, enabling better process understanding and control.

Capacitive Sensor and Alternating Drive Mixing for Microfluidic Applications Using Micro Diaphragm Pumps.

Microfluidic systems are of paramount importance in various fields such as medicine, biology, and pharmacy. Despite the plethora of methods, accurate dosing and mixing of small doses of liquid reagents remain challenges for microfluidics. In this paper, we present a microfluidic device that uses two micro pumps and an alternating drive pattern to fill a microchannel. With a capacitive sensor system, we monitored the fluid process and controlled the micro pumps. In a first experiment, the system was set up to generate a 1:1 mixture between two fluids while using a range of fluid packet sizes from 0.25 to 2 µL and pumping frequencies from 50 to 100 Hz. In this parameter range, a dosing accuracy of 50.3 ± 0.9% was reached, validated by a gravimetric measurement. Other biased mixing ratios were tested as well and showed a deviation of 0.3 ± 0.3% from the targeted mixing ratio. In a second experiment, Trypan blue was used to study the mixing behavior of the system. Within one to two dosed packet sets, the two reagents were reliably mixed. The results are encouraging for future use of micro pumps and capacitive sensing in demanding microfluidic applications.

Extremely Sensitive Microwave Microfluidic Dielectric Sensor Using a Transmission Line Loaded with Shunt LC Resonators.

In this paper, a very high sensitivity microwave-based planar microfluidic sensor is presented. Sensitivity enhancement is achieved and described theoretically and experimentally by eliminating any extra parasitic capacitance not contributing to the sensing mechanism. The sensor consists of a microstrip transmission line loaded with a series connected shunt LC resonator. A microfluidic channel is attached to the area of the highest electric field concentration. The electric field distribution and, therefore, the resonance characteristics are modified by applying microfluidic dielectric samples to the sensing area. The sensor performance and working principle are described through a circuit model analysis. A device prototype is fabricated, and experimental measurements using water/ethanol and water/methanol solutions are presented for validation of the sensing mathematical model.

Soft tubular microfluidics for 2D and 3D applications.

Microfluidics has been the key component for many applications, including biomedical devices, chemical processors, microactuators, and even wearable devices. This technology relies on soft lithography fabrication which requires cleanroom facilities. Although popular, this method is expensive and labor-intensive. Furthermore, current conventional microfluidic chips precludes reconfiguration, making reiterations in design very time-consuming and costly. To address these intrinsic drawbacks of microfabrication, we present an alternative solution for the rapid prototyping of microfluidic elements such as microtubes, valves, and pumps. In addition, we demonstrate how microtubes with channels of various lengths and cross-sections can be attached modularly into 2D and 3D microfluidic systems for functional applications. We introduce a facile method of fabricating elastomeric microtubes as the basic building blocks for microfluidic devices. These microtubes are transparent, biocompatible, highly deformable, and customizable to various sizes and cross-sectional geometries. By configuring the microtubes into deterministic geometry, we enable rapid, low-cost formation of microfluidic assemblies without compromising their precision and functionality. We demonstrate configurable 2D and 3D microfluidic systems for applications in different domains. These include microparticle sorting, microdroplet generation, biocatalytic micromotor, triboelectric sensor, and even wearable sensing. Our approach, termed soft tubular microfluidics, provides a simple, cheaper, and faster solution for users lacking proficiency and access to cleanroom facilities to design and rapidly construct microfluidic devices for their various applications and needs.

Multichannel Electrical Impedance Spectroscopy Analyzer with Microfluidic Sensors.

Impedance spectroscopy is a common approach in assessing passive electrical properties of biological matter. However, several problems appear in microfluidic devices in connection with the requirement for high sensitivity of signal acquisition from small volume sensors. The developed compact and inexpensive analyzer provides impedance spectroscopy measurement from three sensors, both connected in direct and differential modes. Measurement deficiencies are reduced with a novel design of sensors, measurement method, optimized electronics, signal processing, and mechanical design of the analyzer. Proposed solutions are targeted to the creation of reliable point-of-care (POC) diagnostic and monitoring appliances, including lab-on-a-chip type devices in the next steps of development. The test results show the good working ability of the developed analyzer; however, also limitations and problems that require attention and further improvement are appointed.

SAW-Based Phononic Crystal Microfluidic Sensor-Microscale Realization of Velocimetry Approaches for Integrated Analytical Platform Applications.

The current work demonstrates a novel surface acoustic wave (SAW) based phononic crystal sensor approach that allows the integration of a velocimetry-based sensor concept into single chip integrated solutions, such as Lab-on-a-Chip devices. The introduced sensor platform merges advantages of ultrasonic velocimetry analytic systems and a microacoustic sensor approach. It is based on the analysis of structural resonances in a periodic composite arrangement of microfluidic channels confined within a liquid analyte. Completed theoretical and experimental investigations show the ability to utilize periodic structure localized modes for the detection of volumetric properties of liquids and prove the efficacy of the proposed sensor concept.

In Vitro Studies on a Microfluidic Sensor with Embedded Obstacles Using New Antibacterial Synthetic Compounds (1-TDPPO) Mixed Prop-2-en-1-one with Difluoro Phenyl.

This paper describes the use of an analytical microfluidic sensor for accelerating chemo-repellent response and strong anti-bacterial 1-(Thien-2-yl)-3-(2, 6-difluoro phenyl) prop-2-en-1-one (1-TDPPO). The chemically-synthesized antimicrobial agent, which included prop-2-en-1-one and difluoro phenyl groups, was moving through an optically transparent polydimethylsiloxane (PDMS) microfluidic sensor with circular obstacles arranged evenly. The response, growth and distribution of fluorescent labeling Pseudomonas aeruginosa PAO1 against the antimicrobial agent were monitored by confocal laser scanning microscope (CLSM). The microfluidic sensor along with 1-TDPPOin this study exhibits the following advantages: (i) Real-time chemo-repellent responses of cell dynamics; (ii) Rapid eradication of biofilm by embedded obstacles and powerful antibacterial agents, which significantly reduce the response time compared to classical methods; (iii) Minimal consumption of cells and antimicrobial agents; and (iv) Simplifying the process of the normalization of the fluorescence intensity and monitoring of biofilm by captured images and datasets.

The sensing of circRNA with tetrahedral DNA nanostructure modified microfluidic chip.

BACKGROUND: Circular ribonucleic acids (circRNAs) are a type of covalently closed noncoding RNA with disease-relevant expressions, making them promising biomarkers for diagnosis and prognosis. Accurate quantification of circRNA in biological samples is a necessity for their clinical application. So far, methods developed for detecting circRNAs include northern blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), microarray analysis, and RNA sequencing. These methods generally suffer from disadvantages such as large sample consumption, cumbersome process, low selectivity, leading to inaccurate quantification of circRNA. It was thought that the above drawbacks could be eliminated by the construction of a microfluidic sensor. RESULTS: Herein, for the first time, a microfluidic sensor was constructed for circRNA analysis by using tetrahedral DNA nanostructure (TDN) as the skeleton for recognition probes and target-initiated hybridization chain reaction (HCR) as the signal amplification strategy. In the presence of circRNA, the recognition probe targets the circRNA-specific backsplice junction (BSJ). The captured circRNA then triggers the HCR by reacting with two hairpin species whose ends were labeled with 6-FAM, producing long DNA strands with abundant fluorescent labels. By using circ_0061276 as a model circRNA, this method has proven to be able to detect circRNA of attomolar concentration. It also eliminated the interference of linear RNA counterpart, showing high selectivity towards circRNA. The detection process can be implemented isothermally and does not require expensive complicated instruments. Moreover, this biosensor exhibited good performance in analyzing circRNA targets in total RNA extracted from cancer cells. SIGNIFICANCE: This represents the first microfluidic system for detection of circRNA. The biosensor showed merits such as ease of use, low-cost, small sample consumption, high sensitivity and specificity, and good reliability in complex biological matrix, providing a facile tool for circRNA analysis and related disease diagnosis in point-of care application scenes.

Saline based microfluidic soft pressure sensor utilizing a three-dimensional focused electric field for motion and healthcare monitoring.

This paper introduces the 'Spatially Focused Saline-based Pressure Sensor (SF-SaPS)', a novel soft microfluidic pressure sensor featuring a distinctive three-dimensional focusing structure. By critically reducing the cross-sectional area of the microchannel at the focused structure, the SF-SaPS achieves excellent sensitivity to pressure within the sensing region. With the spatially focused region, the SF-SaPS could detect a wide range of pressure from gentle touches to human weight, which is typically unachievable with low-conductivity sensing media such as saline, a medium inherently safe for human use. Beyond its sensitivity, the SF-SaPS exhibits sensing performance and stability comparable with conventional liquid metal-based pressure sensors. Our sensor demonstrated minimal signal drift, a rapid response time of 70 ms under cyclic loading, and 20-day long-term stability tests immersed in water. Additionally, the sensor possesses a transparency advantage unattainable by liquid metal sensors as we utilized transparent polymers and saline. A unique advantage of the SF-SaPS lies in its selective spatial and mechanical sensitivity; as the electrical resistance is highly dependent on changes in the cross-sectional area of the microchannels, the sensor has superior pressure sensitivity compared to bending and strain. Finally, various application examples highlight the SF-SaPS's advantages. By configuring the sensor in a two-axis array, the SF-SaPS facilitates pressure mapping across a plane. Additionally, it proves effective in healthcare monitoring, from radial pulse to finger movements. In conclusion, the SF-SaPS's combination of performance, stability, biocompatibility, and transparency positions this sensor as a versatile tool for applications extending beyond healthcare, as demonstrated in this study.

Single-Ended and Differentially Operated Microwave Microfluidic Sensors for Biomedical Applications.

Background: This research is focused on the design of highly sensitive microfluidic sensors for the applications in liquid dielectric characterizations including biomedical samples. Methods: Considering the narrow-band operation of microfluidic sensors based on microwave resonators, in this study, microfluidic sensors based on the variation of transmission phase in microwave transmission lines (TLs) are proposed. It is shown that among different microwave TLs, slot-lines are an appropriate type of TL for sensing applications because a major portion of the electromagnetic (EM) field passes above the line, where a microfluidic channel can be easily devised. Results: The proposed concept is presented and the functionality of the proposed sensor is validated through full-wave EM simulations. Moreover, the effects of the dimensions of the microfluidic channel and the thickness of the substrate on the sensitivity of the sensor are studied. Furthermore, taking the advantages of differential circuits and systems into account, a differential version of the microfluidic sensor is also presented. It is shown that the sensitivity of the sensor can be adjusted according to the application. Specifically speaking, the sensitivity of the proposed microfluidic sensor is almost linearly proportional to the length of the channel, i.e., the sensitivity can be doubled by doubling the channel length. Conclusions: In this research, it is shown that using slot-line TLs highly sensitive microfluidic sensors can be designed for the applications in liquid dielectric characterizations, especially for biomedical samples where small variations of permittivity have to be detected.

A novel photonic chemosensor for rapidly detecting synthetic dyes in orange juice using colorimetric and spectrophotometric methods.

Synthetic dyes must be monitored and regulated. We aimed to develop a novel photonic chemosensor for rapidly monitoring synthetic dyes based on colorimetric (chemical interactions with optical probes using microfluidic paper-based analytical devices) and UV-Vis spectrophotometric methods. Various types of gold and silver nanoparticles were surveyed to identify the targets. In the presence of silver nanoprisms, the naked eye could visualize the unique and distinctive color changes of Tartrazine (Tar) to green and Sunset Yellow (Sun) to brown; UV-Vis spectrophotometry validated the results. The developed chemosensor showed linear ranges of 0.07-0.3 mM and 0.05-0.2 mM for Tar and Sun, respectively. Sources of interference had minimal effects, confirming the appropriate selectivity of the developed chemosensor. Our novel chemosensor demonstrated excellent analytical performance for measuring Tar and Sun in several types of orange juice as real samples, confirming its incredible potential for use in the food industry.

Application of a fluorescent dye-based microfluidic sensor for real-time detection of mAb aggregates.

The lack of process analytical technologies able to provide real-time information and process control over a biopharmaceutical process has long impaired the transition to continuous biomanufacturing. For the monoclonal antibody (mAb) production, aggregate formation is a major critical quality attribute (CQA) with several known process parameters (i.e., protein concentration and agitation) influencing this phenomenon. The development of a real-time tool to monitor aggregate formation is then crucial to gain control and achieve a continuous processing. Due to an inherent short operation time, miniaturized biosensors placed after each step can be a powerful solution. In this work, the development of a fluorescent dye-based microfluidic sensor for fast at-line PAT is described, using fluorescent dyes to examine possible mAb size differences. A zigzag microchannel, which provides 90% of mixing efficiency under 30 s, coupled to an UV-Vis detector, and using four FDs, was studied and validated. With different generated mAb aggregation samples, the FDs Bis-ANS and CCVJ were able to robustly detect from, at least, 2.5% to 10% of aggregation. The proposed FD-based micromixer is then ultimately implemented and validated in a lab-scale purification system, demonstrating the potential of a miniaturized biosensor to speed up CQAs measurement in a continuous process.

Electrochemical capillary-driven microfluidic DNA sensor for HIV-1 and HCV coinfection analysis.

Electrochemical DNA sensors can be operated in either static or flow-based detection schemes. In static schemes, manual washing steps are still necessary, resulting in a tedious and time-consuming process. In contrast, in flow-based electrochemical sensors, the current response is collected when the solution flows through the electrode continuously. However, the drawback of such a flow system is the low sensitivity due to the limited time for the interaction between the capturing element and the target. Herein, we propose a novel electrochemical capillary-driven microfluidic DNA sensor to combine the advantages of static and flow-based electrochemical detection systems into a single device by incorporating burst valve technology. The microfluidic device with a two-electrode configuration was applied for the simultaneous detection of two different DNA markers, human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) cDNA, via the specific interaction between pyrrolidinyl peptide nucleic acids (PNA) probes and the DNA target. The integrated system, while requiring a small sample volume (7 µL for each sample loading port) and less analysis time, achieved good performance in terms of the limits of detection (LOD) (3SDblank/slope) and quantification (LOQ) (10SDblank/slope) at 1.45 nM and 4.79 nM for HIV and 1.20 nM and 3.96 nM for HCV, respectively. The simultaneous detection of HIV-1 and HCV cDNA prepared from human blood samples showed results that are in complete agreement with the RT‒PCR assay. The results qualify this platform as a promising alternative for the analysis of either HIV-1/HCV or coinfection that can be easily adapted for other clinically important nucleic acid-based markers.

CuO Nanozymes as Multifunctional Signal Labels for Efficiently Quenching the Photocurrent of ZnO/Au/AgSbS2 Hybrids and Initiating a Strong Fluorescent Signal in a Dual-Mode Microfluidic Sensing Platform.

A novel dual-mode microfluidic sensing platform based on CuO nanozymes as a photoelectrochemical (PEC)-fluorescent (FL) multifunctional signal label was developed for ultrasensitive neuron specific enolase (NSE) detection. Herein, ZnO/Au/AgSbS2 hybrids, possessing excellent PEC properties, were first exploited as a sensing matrix to provide a stable photocurrent. The controlled synthesis of photoactive ZnO nanoflowers (NFs) was successfully conducted using a microfluidic reactor in the scale of seconds. Furthermore, the photocurrent of ZnO NFs decorated by Au and AgSbS2 nanoparticles significantly improved, owing to the local surface plasma resonance effect of Au and matching band structure between ZnO and AgSbS2. A strategy of catalytic oxidation ascorbic acid (AA) by CuO nanozymes was proposed to quench the PEC signals and initiate FL signals. CuO nanoparticles growing on conductive carbon spheres (CuO@CSs) as secondary antibodies' labels could efficiently catalyze the oxidation of AA to achieve a PEC "signal-off" state. Then, the produced dehydroascorbic acid reacting with o-phenylenediamine opportunely generated a strong FL signal. Importantly, wide linear ranges of 0.0001-150 ng/mL for the PEC technique and 0.001-150 ng/mL for the FL method with a low detection limit of 0.028 and 0.25 pg/mL, respectively, could guarantee the sensitive detection of NSE.

Integrated electrochemical microfluidic sensor with hierarchically porous nanoarrays modified graphene fiber microelectrode for bioassay.

The development of high-efficient biosensing systems for rapid and sensitive detection of disease-related biomarkers in human samples is of great significance for disease diagnosis and treatment in clinical practice. In this work, we develop an integrated electrochemical microfluidic sensing platform based on freestanding graphene fiber (GF) microelectrode for bioassay. In order to improve the electrocatalytic activity of GF microelectrode, it has been modified by unique 3D well-ordered hierarchically porous nickel-cobalt phosphide (NiCoP) nanosheet arrays (NSAs). Benefiting from the excellent electrochemical properties and structural merits, the resultant NiCoP-NSAs modified GF microelectrode shows excellent sensing performances towards neurotransmitter dopamine (DA), with a high sensitivity of 5.56 µA cm-2 µM-1, a low detection limit of 14 nM, as well as good selectivity, reproducibility and stability. Furthermore, in virtue of the miniaturized size and good mechanical properties, the nanohybrid GF microelectrode can be embedded into a home-made microfluidic chip to construct an integrated electrochemical microfluidic sensing device, which has been used for sensitive analysis of DA in minimal volume of human serum and urine samples, and in situ tracking DA released from neuroblastoma cells SHSY-5Y under the stimulation for physio-pathological and pharmacological study of nervous system-related diseases.

All-Serotype Dengue Virus Detection through Multilayered Origami-Based Paper/Polymer Microfluidics.

The dengue virus (DENV) infection commonly triggers threatening seasonal outbreaks all around the globe (estimated yearly infections are in the order of 100 million, combining all the viral serotypes), testifying the need for early detection to facilitate disease management and patient recovery. The laboratory-based testing procedures for detecting DENV infection early enough are challenged by the need of resourced settings that result in inevitable cost penalty and unwarranted delay in obtaining the test results due to distance-related factors with respect to the patient's location. Recognizing that the introduction of alternative extreme point-of-care technologies for early detection may potentially mitigate this challenge largely, we develop here a multiplex paper/polymer-based detection strip that interfaces with an all-in-one simple portable device, synchronizing the pipeline of nucleic acid isolation, isothermal amplification, and colorimetric analytics as well as readout for detecting all the four serotypes of dengue viruses in around 30 min from about 50 µL of human blood serum with high specificity and sensitivity. Aligned with the mandatory guidelines of the World Health Organization, the ultralow-cost test is ideal for dissemination at different community centers via a user-friendly device interface, not only as a critical surveillance measure in recognizing the potential cocirculation of the infection across regions that are hyperendemic for all four DENV serotypes but also for facilitating effective monitoring of patients infected by any one of the particular viral serotypes as well as timely administration of life-saving measures on need.

Microfluidic sensor integrated with nanochannel liquid conjunct Ag/AgCl reference electrode for trace Pb(II) measurement.

Pollution due to heavy metals is becoming increasingly hazardous; therefore, demand for the large-scale deployment of sensor nodes for water quality monitoring has increased. The development of integrated and miniaturised sensors for detecting heavy metals is necessary. Herein, an integrated microfluidic sensor based on a "glass-silicon-glass" sandwich structure is proposed for Pb2+ detection. This micro-sensor consists of a nanochannel liquid conjunct Ag/AgCl reference electrode(RE), a working electrode with a three-dimensional Au micropillar array, and a detection chamber for sample measurement. The potential fluctuation of the RE in this sensor was only 0.62% over seven days, remaining relatively stable. Under optimal conditions, the limit of detection and sensitivity for lead were 0.13 µg L-1 (S/N = 3) and 52.30 nA (µg L-1)-1, respectively. The linearity of the sensor for detecting lead was good in the concentration range of 0.50-150 µg L-1 (R2 = 0.9989). Moreover, the proposed microsensor showed high selectivity for Pb2+ and achieved sensitive detection of trace Pb2+ in different water samples. Therefore, this integrated and miniaturised sensor is a practical tool for trace lead detection, allowing the development of large scale sensor network for water monitoring.

Detection of Ca2+-induced acetylcholine released from leukemic T-cells using an amperometric microfluidic sensor.

A microfluidic structured-dual electrodes sensor comprising of a pair of screen printed carbon electrodes was fabricated to detect acetylcholine, where one of them was used for an enzyme reaction and another for a detection electrode. The former was coated with gold nanoparticles and the latter with a porous gold layer, followed by electropolymerization of 2, 2:5,2-terthiophene-3-(p-benzoic acid) (pTTBA) on both the electrodes. Then, acetylcholinesterase was covalently attached onto the reaction electrode, and hydrazine and choline oxidase were co-immobilized on the detection electrode. The layers of both modified electrodes were characterized employing voltammetry, field emission scanning electron microscopy, X-ray photoelectron spectroscopy, and quartz crystal microscopy. After the modifications of both electrode surfaces, they were precisely faced each other to form a microfluidic channel structure, where H2O2 produced from the sequential enzymatic reactions was reduced by hydrazine to obtain the analytical signal which was analyzed by the detection electrode. The microfluidic sensor at the optimized experimental conditions exhibited a wide dynamic range from 0.7nM to 1500µM with the detection limit of 0.6 ± 0.1nM based on 3s (S/N = 3). The biomedical application of the proposed sensor was evaluated by detecting acetylcholine in human plasma samples. Moreover, the Ca2+-induced acetylcholine released in leukemic T-cells was also investigated to show the in vitro detection ability of the designed microfluidic sensor. Interference due to the real component matrix were also studied and long term stability of the designed sensor was evaluated. The analytical performance of the designed sensor was also compared with commercially available ACh detection kit.

Self-Powered Triboelectric Micro Liquid/Gas Flow Sensor for Microfluidics.

Liquid and gas flow sensors are important components of the micro total analysis systems (µTAS) for modern analytical sciences. In this paper, we proposed a self-powered triboelectric microfluidic sensor (TMS) by utilizing the signals produced from the droplet/bubble via the capillary and the triboelectrification effects on the liquid/solid interface for real-time liquid and gas flow detection. By alternating capillary with different diameters, the sensor's detecting range and sensitivity can be adjusted. Both the relationship between the droplet/bubble and capillary size, and the output signal of the sensor are systematically studied. By demonstrating the monitoring of the transfusion process for a patient and the gas flow produced from an injector, it shows that TMS has a great potential in building a self-powered micro total analysis system.

A mini-review on functional nucleic acids-based heavy metal ion detection.

Recent years have witnessed great progress in developing functional nucleic acids (FNAs)-based sensors for the detection of heavy metal ion. In this review, four types of the FNAs that most widely-used in heavy metal ions detection were briefly introduced and a dozen of recently published review articles which summarized those FNAs-based sensors were introduced. Particularly, according to the degree of automation and system integration, those FNAs-based sensors which belong to the lab-on-a-chip (LOC) category were reviewed in more detail by classifying them into six types such as microfluidic LOC system, microchip, lateral flow dipstick, personal glucose meter, microfluidic paper-based analytical devices (µPADs) and disc-based analytical platform. After gave a brief description of the sensing strategies, properties, advantages or disadvantages of these FNAs-based sensors, existing problems and future perspectives were also discussed.