Self-Powered Microfluidics for Point-of-Care Solutions: From Sampling to Detection of Proteins and Nucleic Acids.

Self-powered microfluidics presents a revolutionary approach to address the challenges of healthcare in decentralized and point-of-care settings where limited access to resources and infrastructure prevails or rapid clinical decision-making is critical. These microfluidic systems exploit physical and chemical phenomena, such as capillary forces and surface tension, to manipulate tiny volumes of fluids without the need for external power sources, making them cost-effective and highly portable. Recent technological advancements have demonstrated the ability to preprogram complex multistep liquid operations within the microfluidic circuit of these standalone systems, which enabled the integration of sensitive detection and readout principles. This chapter first addresses how the accessibility to in vitro diagnostics can be improved by shifting toward decentralized approaches like remote microsampling and point-of-care testing. Next, the crucial role of self-powered microfluidic technologies to enable this patient-centric healthcare transition is emphasized using various state-of-the-art examples, with a primary focus on applications related to biofluid collection and the detection of either proteins or nucleic acids. This chapter concludes with a summary of the main findings and our vision of the future perspectives in the field of self-powered microfluidic technologies and their use for in vitro diagnostics applications.

Revolutionizing sample preparation: a novel autonomous microfluidic platform for serial dilution.

Dilution is a standard fluid operation widely employed in the sample preparation process of many bio(chemical) assays. It serves multiple essential functions such as sample mixing with certain reagents at specific dilution ratios, reducing sample matrix effects, bringing target analytes within the linear assay detection range, among many others. Traditionally, sample processing is performed in laboratory settings through manual or automated pipetting. When working in resource-limited settings, however, neither trained personnel nor proper laboratory equipment are available limiting the accessibility to high-quality diagnostic tests. In this work, we present a novel standalone and fully automated microfluidic platform for the stepwise preparation of serial dilutions without the need for any active elements. Stepwise dilution is achieved using the coordinated burst action of hydrophobic burst valves to first isolate a precisely metered volume from an applied sample drop and subsequently merge it with a prefilled diluent liquid. Downstream, expansion chambers are used to mix both reagents into a homogeneous solution. The dilution module was characterized to generate accurate and reproducible (CV < 7%) dilutions for targeted dilution factors of 2, 5 and 10×, respectively. Three dilution modules were coupled in series to generate three-fold logarithmic (log5 or log10) dilutions, with excellent linearity (R2 > 0.99). Its compatibility with whole blood was furthermore illustrated, proving its applicability for automating and downscaling bioassays with complex biological matrices. Finally, autonomous on-chip serial dilution was demonstrated by incorporating the self-powered (i)SIMPLE technology as a passive driving source for liquid manipulation. We believe that the simplicity and modularity of the presented autonomous dilution platform are of interest to many point-of-care applications in which sample dilution and reagent mixing are of importance.

SeParate: multiway fluorescence-activated droplet sorting based on integration of serial and parallel triaging concepts.

Fluorescence-activated droplet sorting (FADS) has emerged as a versatile high-throughput sorting tool that is, unlike most fluorescence-activated cell sorting (FACS) platforms, capable of sorting droplet-compartmentalized cells, cell secretions, entire enzymatic reactions and more. Recently, multiplex FADS platforms have been developed for the sorting of multi-fluorophore populations towards different outlets in addition to the standard, more commonly used, 2-way FADS platform. These multiplex FADS platforms consist of either multiple 2-way junctions one after the other (i.e. serial sorters) or of one junction sorting droplets in more than 2 outlets (i.e. parallel sorters). In this work, we present SeParate, a novel platform based on integrating s̲e̲rial and p̲a̲r̲allel sorting principles for accura̲t̲e̲ multiplex droplet sorting that is able to mitigate limitations of current multiplex sorters. We show the SeParate platform and its capability in highly accurate 4-way sorting of a multi-fluorophore population into four subpopulations with the potential to expand to more. More specifically, the SeParate platform was thoroughly validated using mixed populations of fluorescent beads and picoinjected droplets, yielding sorting accuracies up to 100% and 99.9%, respectively. Finally, transfected HEK-293T cells were sorted employing two different optical setups, resulting in an accuracy up to 99.5%. SeParate's high accuracy for a diverse set of samples, including highly variable biological specimens, together with its scalability beyond the demonstrated 4-way sorting, warrants a broad applicability for multi-fluorophore studies in life sciences, environmental sciences and others.

Innovative Fabrication of Hollow Microneedle Arrays Enabling Blood Sampling with a Self-Powered Microfluidic Patch.

Microneedles are gaining a lot of attention in the context of sampling cutaneous biofluids such as capillary blood. Their minimal invasiveness and user-friendliness make them a prominent substitute for venous puncture or finger-pricking. Although the latter is suitable for self-sampling, the impracticality of manual handling and the difficulty of obtaining enough qualitative sample is driving the search for better solutions. In this context, hollow microneedle arrays (HMNAs) are particularly interesting for completely integrating sample-to-answer solutions as they create a duct between the skin and the sampling device. However, the fabrication of sharp-tipped HMNAs with a high aspect ratio (AR) is challenging, especially since a length of ≥1500 µm is desired to reach the blood capillaries. In this paper, we first described a novel two-step fabrication protocol for HMNAs in stainless steel by percussion laser drilling and subsequent micro-milling. The HMNAs were then integrated into a self-powered microfluidic sampling patch, containing a capillary pump which was optimized to generate negative pressure differences up to 40.9 ± 1.8 kPa. The sampling patch was validated in vitro, showing the feasibility of sampling 40 µL of liquid. It is anticipated that our proof-of-concept is a starting point for more sophisticated all-in-one biofluid sampling and point-of-care testing systems.

Bridging the Gap between Digital Assays and Point-of-Care Testing: Automated, Low Cost, and Ultrasensitive Detection of Thyroid Stimulating Hormone.

Medical diagnostics is moving toward disease-related target detection at very low concentrations because of the (1) quest for early-stage diagnosis, at a point where only limited target amounts are present, (2) trend toward minimally invasive sample extraction, yielding samples containing low concentrations of target, and (3) need for straightforward sample collection, usually resulting in limited volume collected. Hence, diagnostic tools allowing ultrasensitive target detection at the point-of-care (POC) are crucial for simplified and timely diagnosis of many illnesses. Therefore, we developed an innovative, fully integrated, semi-automated, and economically viable platform based on (1) digital microfluidics (DMF), enabling automated manipulation and analysis of very low sample volumes and (2) low-cost disposable DMF chips with microwell arrays, fabricated via roll-to-roll processes and allowing digital target counting. Thyroid stimulating hormone detection was chosen as a relevant application to show the potential of the system. The assay buffer was selected using design of experiments, and the assay was optimized in terms of reagent concentration and incubation time toward maximum sensitivity. The hydrophobic-in-hydrophobic microwells showed an unparalleled seeding efficiency of 97.6% ± 0.6%. A calculated LOD of 0.0013 µIU/mL was obtained, showing the great potential of the platform, especially taking into account the very low sample volume analyzed (1.1 µL). Although validation (in biological matrix) and industrialization (full automation) steps still need to be taken, it is clear that the combination of DMF, low-cost DMF chips, and digital analyte counting in microwell arrays enables the implementation of ultrasensitive and reliable target detection at the POC.

Next generation point-of-care test for therapeutic drug monitoring of adalimumab in patients diagnosed with autoimmune diseases.

Therapeutic drug monitoring (TDM) of adalimumab (ADM) at the point-of-care (POC) is key to prevent loss of response but has not been accomplished to date because true POC testing solutions are still lacking. Here, we present a novel "whole blood in - result out" self-powered microfluidic chip for detecting ADM within 30 min to enable TDM at POC. Hereto, we first demonstrated on-chip plasma separation from whole blood, followed by downscaling an ADM ELISA with maintained specificity and sensitivity in plasma. This assay was then performed on a robust and easy-to-use microfluidic chip we designed based on (i)SIMPLE technology, allowing autonomous function upon single finger press activation, which was successfully validated with patient samples. Herein, we prove the potential of our technology to detect targets starting from whole blood introduced directly on-chip and to integrate various immunoassays, both for TDM and other in vitro diagnostics applications, like infectious diseases.

Point-of-care therapeutic drug monitoring of adalimumab by integrating a FO-SPR biosensor in a self-powered microfluidic cartridge.

Disease treatment with advanced biological therapies such as adalimumab (ADM), although largely beneficial, is still costly and suffers from loss of response. To tackle these aspects, therapeutic drug monitoring (TDM) is proposed to improve treatment dosing and efficacy, but is often associated with long sampling-to-result workflows. Here, we present an in-house constructed ADM-sensor, allowing TDM of ADM at the doctor's office. This biosensor brings fiber optic surface plasmon resonance (FO-SPR), combined with self-powered microfluidics, to a point of care (POC) setting for the first time. After developing a rapid FO-SPR sandwich bioassay for ADM detection on a commercial FO-SPR device, this bioassay was implemented on the fully-integrated ADM-sensor. For the latter, we combined (I) a gold coated fiber optic (FO) probe for bioassay implementation and (II) an FO-SPR readout system with (III) the self-powered iSIMPLE microfluidic technology empowering plasma sample and reagent mixing on the-cartridge as well as connection to the FO-SPR readout system. With a calculated limit of detection (LOD) of 0.35 µg/mL in undiluted plasma, and a total time-to-result (TTR) within 12 min, this innovative biosensor demonstrated a comparable performance to existing POC biosensors for ADM quantification in patient plasma samples, while requiring only 1 µL of plasma. Whereas this study demonstrates great potential for FO-SPR biosensing at the POC using ADM as a model case, it also shows huge potential for bedside TDM of other drugs (e.g. other immunosuppressants, anti-epileptics and antibiotics), as the bioassay is highly amenable to adaptation.

Precise sample metering method by coordinated burst action of hydrophobic burst valves applied to dried blood spot collection.

Dried blood spot (DBS) sampling by finger-pricking has recently gained a lot of interest as an alternative sample collection method. The reduced invasiveness, requirement of lower sample volumes and suitability for long-term storage at room temperature make DBS ideal for use in home settings or low-resource environments. However, traditional protocols often suffer from biased analysis data due to variable and not exactly known blood volumes present in the samples. In this work, a novel device has been developed to split-off precisely metered volumes from a blood drop and load them on pre-cut filter paper. Hereto, hydrophobic burst valves (HBV) were developed to temporarily retain a fluid flow, configurable to burst at pressures within a range of 175-600 Pa. By combining HBVs with different burst pressures, a volume metering system was developed to allow parallel metering of multiple pre-defined sample volumes. The system was shown to be accurate and consistent for blood volumes between 5-15 µL and for hematocrit levels spanning the range of 25-70%. Finally, a point-of-care DBS sampling device was developed combining the self-powered microfluidic SIMPLE technology. To evaluate the system's practical applicability, a validation study in the context of therapeutic drug monitoring of biologicals was performed using adalimumab-spiked blood samples. Microfluidic DBS samples showed good performance compared to the traditional DBS method with improved recovery rates (86% over 62%). This innovative metering system, allowing for parallelization and integration with complex liquid manipulations, will greatly impact the field of robust sampling, sample preparation, storage and analysis at the point-of-care.

Innovative Hydrophobic Valve Allows Complex Liquid Manipulations in a Self-Powered Channel-Based Microfluidic Device.

We present an innovative, simple, and versatile hydrophobic valve enabling all-important complex liquid manipulations on self-powered, channel-based microfluidic devices and as such being extremely valuable for the design of highly demanding point-of-care (POC) platforms. The presented hydrophobic valve is made of filter paper treated with a fluorinated compound (i.e., Aquapel) and shows both superhydrophobic properties (contact angle up to 155°) and high resistance to liquid pressure (up to 9 kPa), while retaining gas permeability and utter fabrication simplicity. Whereas this valve can be integrated in any channel-based system and can be used both as a vent, to delay liquid displacement on chip, or as a barrier, to stop the liquid flow in a certain direction, in this work we demonstrate some of its capacities by combining it with our in house developed self-powered SIMPLE and iSIMPLE platforms. First, we integrated it with the infusion iSIMPLE pump, thus generating completely fail-proof activation regardless of how the operator is actuating the system. Second, we used hydrophobic valves as both barrier and vent in the same microfluidic chip, which allowed the combination of two SIMPLE pumps for splitting one sample in two parallel channels. This attribute is fundamental for achieving multiplex analysis on completely autonomous microfluidic platforms. Finally, we achieved an unprecedented liquid manipulation for a self-powered microfluidic platform, namely, shuttling of liquid, after a single user activation by combining for the first time SIMPLE and iSIMPLE with the developed hydrophobic vent and barrier, all in a single chip. These results convincingly demonstrated that the developed hydrophobic valve combined with SIMPLE/iSIMPLE presents an essential building block for an ideal POC system, which is self-powered, inexpensive, and robust and can perform complex bioassays upon a single user activation.

Self-powered infusion microfluidic pump for ex vivo drug delivery.

In this work, we present a new iSIMPLE concept (infusion Self-powered Imbibing Microfluidic Pump by Liquid Encapsulation), which requires no external power for activation nor liquid manipulation, it is easy to use while its fabrication method is extremely simple, inexpensive and suited for mass replication. The pump consists of a working liquid, which is - after finger activation - absorbed in a porous material (e.g. filter paper). The air expelled from the porous material increases the pressure in the downstream outlet channel and propels the outlet liquid (i.e. the sample) through the channel or ejects it. Here we investigated the influence of different filter papers on the iSIMPLE flow rates, achieving a wide range from 30 down to 0.07 µL/min. We also demonstrated the versatility of the iSIMPLE in terms of the liquid volume that can be manipulated (from 0.5 µL up to 150 µL) and the working pressure reaching 64 kPa, unprecedented high for a self-powered microfluidics pump. In addition, using a 34 G microneedle mounted on the iSIMPLE, we successfully injected liquids with different viscosities (from 0.93 up to 55.88 cP) both into an agarose matrix and a skin-like biological ex vivo substrate (i.e. chicken breast tissue). This work validated the compatibility of the iSIMPLE with drug delivery in a controlled way into a skin-like matrix, envisioning a whole new scenario for intradermal injections using self-contained skin patch. In addition, due to the extreme flexibility of the design and manufacturing, the iSIMPLE concept offers enormous opportunities for completely autonomous, portable and cost effective LOC devices.

Creasensor: SIMPLE technology for creatinine detection in plasma.

The lab-on-a-chip (LOC) field has witnessed an excess of new technology concepts, especially for the point-of-care (POC) applications. However, only few concepts reached the POC market often because of challenging integration with pumping and detection systems as well as with complex biological assays. Recently, a new technology termed SIMPLE was introduced as a promising POC platform due to its features of being self-powered, autonomous in liquid manipulations, cost-effective and amenable to mass production. In this paper, we improved the SIMPLE design and fabrication and demonstrated for the first time that the SIMPLE platform can be successfully integrated with biological assays by quantifying creatinine, biomarker for chronic kidney disease, in plasma samples. To validate the robustness of the SIMPLE technology, we integrated a SIMPLE-based microfluidic cartridge with colorimetric read-out system into the benchtop Creasensor. This allowed us to perform on-field validation of the Creasensor in a single-blind study with 16 plasma samples, showing excellent agreement between measured and spiked creatinine concentrations (ICC: 0.97). Moreover, the range of clinically relevant concentrations (0.76-20 mg/dL), the sample volume (5 µL) and time-to-result of only 5 min matched the Creasensor performance with both lab based and POC benchmark technologies. This study demonstrated for the first time outstanding robustness of the SIMPLE in supporting the implementation of biological assays. The SIMPLE flexibility in liquid manipulation and compatibility with different sample matrices opens up numerous opportunities for implementing more complex assays and expanding its POC applications portfolio.