Complex Bioassays Research Articles

Programmable droplet microfluidics for complex multistep bioassays

AbstractProgrammable droplet microfluidics (PDM) refers to a microfluidic device that integrates the functionality of a series of droplets with distinct spatial locations in a designated temporal order. PDM streamlines the intricate workflow of complex bioassays by enabling programmable and macroscopic droplet displacements, in which the droplets serve as reservoirs for reagents, microvalves for liquid insulation, and in some cases micropumps for mass transportation. As these droplets are intangible structures, the need for expensive microfabrication procedures is eliminated. Furthermore, the parallelization of the droplet series provides flexibility in controlling the throughput of the microfluidic analysis system. This paper provides a comprehensive review of PDMs enabled by various microfluidic mechanisms, including magnetism actuation, relative liquid displacement, capillary suction and sequential microdisplacement. Additionally, important applications of PDM systems for nucleic acid detection, immunoassay, drug testing, and sample recovery are also introduced. In conclusion, PDM demonstrates its potential as a highly advantageous tool for executing intricate multistep bioassays on a microfluidic platform. These technologies have exhibited superiority over their traditional counterparts in terms of size reduction, automation, and low sample/reagent consumption.

An Efficient Capacity-based Routing Technique on MEDA-based Biochips

Micro Electrodes Dot Array (MEDA) based biochips are emerging microdroplets handling technology that empowers supervised fluids governance on a set of microelectrodes. The MEDA-based architecture enables the dynamic routing of droplets with variable sizes for better accuracy and convenience. Efficient droplet routing with prevention of cross-contamination and maintaining all fluidic restriction is the key challenging area on the MEDA-based biochips. We use a two-stage method that involves global and detailed routing. In order to determine the route capacity of every feasible routing path for each droplet, a graph denoting the capacity has been created during the global routing phase. The proposed method assigns the priority of each net depending on the influence factors of the other nets and blockages present in the bioassay. The proposed approach enables 80% of droplet routing without any cross-contamination, based on the First Priority Movement (FPM) and Second Priority Movement (SPM) in the first stage. A capacity graph provides maximum droplets flow using FPM and finds the suitable channel to reshape the size of the droplets to avoid collision on the routing paths to accomplish 100% routing. Experimental results demonstrate that even in complex bio-assays, our proposed phase-wise router reduces 20% of the latest arrival time (T la) and minimizes 9% average time (T avg) by decreasing 15% of microelectrodes count compared to other routing protocols.

A deep-reinforcement learning approach for optimizing homogeneous droplet routing in digital microfluidic biochips

Over the past two decades, digital microfluidic biochips have been in much demand for safety-critical and biomedical applications and increasingly important in point-of-care analysis, drug discovery, and immunoassays, among other areas. However, for complex bioassays, finding routes for the transportation of droplets in an electrowetting-on-dielectric digital biochip while maintaining their discreteness is a challenging task. In this study, we propose a deep reinforcement learning-based droplet routing technique for digital microfluidic biochips. The technique is implemented on a distributed architecture to optimize the possible paths for predefined source–target pairs of droplets. The actors of the technique calculate the possible routes of the source–target pairs and store the experience in a replay buffer, and the learner fetches the experiences and updates the routing paths. The proposed algorithm was applied to benchmark suites I and III as two different test benches, and it achieved significant improvements over state-of-the-art techniques.

High-Resolution Patterning of Hydrogel Sensing Motifs within Fibrous Substrates for Sensitive and Multiplexed Detection of Biomarkers.

There has been an increasing and urgent demand to develop nucleic acid bioassays which not only offer high analytical performance but which are also amenable with point-of-care testing. Hydrogels present a versatile class of materials with biocompatible antifouling properties and the ability to be engineered for a range of advanced sensing applications. Fibrous substrates like nitrocellulose offer low-cost and durable platforms to run complex bioassays while enabling portability and ease of handling. We demonstrate herein the ability to synergistically combine these two materials into a portable biosensing platform by leveraging projection lithography. We demonstrate the direct polymerization of hydrogel sensing motifs within a range of fibrous substrates with precise control over their shape, size, location, and functionality. Spatial encoding of the hydrogel motifs enables the multiplex detection of multiple biomarkers on the same test. As a proof-of-concept, we apply the platform to the detection of microRNA, an emerging class of circulating biomarkers with promising potential for early diagnosis and monitoring of cancer. The assay offers a large dynamic range (over three orders of magnitude), high sensitivity (limit of detection of 2.5 amol), as well as versatility and ease of handling. Finally, the bioassay is validated using real biological samples, namely, total RNA extracted from the sera of late-stage breast cancer patients, demonstrating its utility and compatibility with clinical biosensing applications.

High-Throughput Label-Free Enzymatic Assays Using Desorption Electrospray-Ionization Mass Spectrometry.

High-throughput (HT) enzymatic assays, which typically rely on labeled compounds and plate readers, are important for drug discovery. Mass spectrometry (MS) provides an alternative method of performing HT label-free assays. Here we demonstrate the use of a HT platform based on desorption electrospray ionization (DESI) MS for the label-free study of enzymatic reactions directly from the bioassay matrix with an effective analysis time of 0.3 s per sample. This system allows for thorough analysis of the enzymatic process through monitoring of its substrate and product after an external calibration. We show the platform capabilities by an in-depth study of the acetylcholinesterase assay, including kinetic parameter determination, rapid inhibitor screening, and further characterization of positive hits (that is, IC50 and Ki ), as well as inhibition-reactivation assays. We anticipate that the expansion of this platform has high potential impact in label-free enzymology as well as in drug discovery.

Allosteric DNA molecular beacons: Using a novel mechanism to develop universal biosensor arrays to fully discriminate DNA/RNA analogues

With a unique stem-loop hairpin structure, DNA molecular beacons have proven effective in the accurate discrimination of nucleic acid sequences with small changes. However, there are still many problems in current molecular beacons such as stringent assay conditions, insufficient specificity, non-universality and difficulty in multi-targets recognition. One universal type of allosteric DNA molecular beacons was developed in this work by separating the recognition element and signal reporter. The response process was demonstrated by real-time fluorescence simulations and native gel electrophoresis. Used as universal elements in biosensor arrays, the allosteric DNA molecular beacons can fully discriminate various muted DNA/RNA targets as separate clusters without any overlap. The new allosteric DNA molecular beacons have many advantages over conventional ones, typically used for biosensor arrays, and expand the application prospect of molecular beacons for complex bioassay tasks.

On-board control of wax valve on active centrifugal microfluidic chip and its application for plasmid DNA extraction

For the realization of bioassay with complex fluidic manipulation and logic operation on lab-on-a-disc platform, we present an active integrated centrifugal microfluidic chip based on the on-board control of wax valves within a multilayer complex chip. The multilayer hybrid structure including a microfluidic layer and a printing circuit board (PCB) layer utilizes the digital logic of electronic system to control the logic of liquid flow in microfluidic layer. The coupling mechanism between both layers is based on heat transfer, namely, the heating resistors in PCB layer are used to melt and open the paraffin wax valves in microfluidic layer. Without the limitation of surface tension-dependent valves, the application of active valve could be freely designed, which can largely extend the ability of integration on microfluidic chip. Many complex functional units including liquid sequential loading and switching of liquid flow are demonstrated. As an application, we also present a multilayer complex chip for plasmid DNA extraction based on our platform. In a word, our active centrifugal microfluidic platform provides a solution for the integration of complex bioassay on rotating disc, which has great potential in the applications of point-of-care diagnostics (POC).

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.

Biotesting of Water Contaminated by Microorganisms

This study shows the possibility of applying the complex bioassay of water to determine the degree of its toxicity under conditions of microbiological contamination. The toxic effect of microorganisms (Candida albicans) at their density 1 × 102 CFU/cm3 was detected on fish cells using the micronucleus test method. The possible mechanisms of toxic effect of enhanced microbial contamination of water on animal and plant test-organisms have been studied.

Active droplet-array (ADA) microfluidics enables multiplexed complex bioassays for point of care testing.

We introduce a novel and versatile microfluidic technology that allows parallel and multi-step bioanalytical procedures to be simply implemented by switching reagent-containing droplet arrays among alternative interaction zones for intended mass or energy transport in a programmable manner. This enables multiplexed complex bioassays for point-of-care testing.

Cytogenotoxicity of shakes added with synthetic microingredients

The goal of this study was to evaluate the cytotoxicity and genotoxicity of diet shakes of three brands widely marketed in Brazil, named in this study of A, B and C. Assessments were carried out on root meristem cells of Allium cepa at exposure times 24 and 48 hours. The products (treatments) for A and B were evaluated at concentrations of 0.05; 0.10 and 0.20 g/mL, and C at the concentration 0.08; 0.16 and 0.32 g/mL. The diet products at the three concentrations tested, including those suggested as ideal for consumption by the manufacturers, at the two exposure times considered, were found to have significant antiproliferative effect on the meristems evaluated. However, none of the treatments caused significant cellular changes to the tissues of roots. Thus, the products tested were cytotoxic, but not genotoxic, to root meristem cells of A. cepa. Furthermore, it was observed that in all the treatments performed, in the three concentrations, the majority of the dividing cells were at prophase, not being observed cells at anaphase and telophase. Thus, it is suggested to conduct more in-depth studies on the cytotoxicity of these shakes in physiologically more complex bioassays for the verification and deepening of the data obtained here.

A valveless capillary mixing system using a novel approach for passive flow control

Using capillary-driven forces as a means to pump and actuate fluids is attractive for many applications including portable, lab-on-a-chip diagnostic systems. However, the lack of reliable and easy-to-integrate means of flow control remains a major challenge for implementing more complex bioassays using capillary microfluidics. This paper presents an easy-to-fabricate, valveless capillary system allowing for high-reliability flow control without using traditional capillary stop or trigger valves. Analytical calculations were used to deduce a design criterion for flow control in the valveless system. Furthermore, electrical analogy modeling was used to analyze and optimize the flow characteristics in the valveless system. Silicon-based test structures and capillary mixing systems were fabricated to check the design criterion and benchmark the electrical analogy model, respectively. Experimental results were in good agreement for the analytically derived flow control criterion. Experimental measurements of the liquid–vapor interface position with respect to time agreed well with the electrical analogy modeling results. The flow control scheme developed in the work provides designers with another means to realize complex fluid processing requirements without the need for traditional valve structures.

Microfluidics as a new tool in radiation biology

Ionizing radiations interact with molecules at the cellular and molecular levels leading to several biochemical modifications that may be responsible for biological effects on tissue or whole organisms. The study of these changes is difficult because of the complexity of the biological response(s) to radiations and the lack of reliable models able to mimic the whole molecular phenomenon and different communications between the various cell networks, from the cell activation to the macroscopic effect at the tissue or organismal level. Microfluidics, the science and technology of systems that can handle small amounts of fluids in confined and controlled environment, has been an emerging field for several years. Some microfluidic devices, even at early stages of development, may already help radiobiological research by proposing new approaches to study cellular, tissue and total-body behavior upon irradiation. These devices may also be used in clinical biodosimetry since microfluidic technology is frequently developed for integrating complex bioassay chemistries into automated user-friendly, reproducible and sensitive analyses. In this review, we discuss the use, numerous advantages, and possible future of microfluidic technology in the field of radiobiology. We will also examine the disadvantages and required improvements for microfluidics to be fully practical in radiation research and to become an enabling tool for radiobiologists and radiation oncologists.

Caenorhabditis elegans-based Model Systems for Antifungal Drug Discovery

The substantial morbidity and mortality associated with invasive fungal infections constitute undisputed tokens of their severity. The continued expansion of susceptible population groups (such as immunocompromised individuals, patients undergoing extensive surgery, and those hospitalized with serious underlying diseases especially in the intensive care unit) and the limitations of current antifungal agents due to toxicity issues or to the development of resistance, mandate the development of novel antifungal drugs. Currently, drug discovery is transitioning from the traditional in vitro large-scale screens of chemical libraries to more complex bioassays, including in vivo studies on whole animals; invertebrates, such as Caenorhabditis elegans, are thus gaining momentum as screening tools. Key pathogenesis features of fungal infections, including filament formation, are expressed in certain invertebrate and mammalian hosts; among the various potential hosts, C. elegans provides an attractive platform both for the study of host-pathogen interactions and the identification of new antifungal agents. Advantages of compound screening in this facile, relatively inexpensive and not as ethically challenged whole-animal context, include the simultaneous assessment of antifungal efficacy and toxicity that could result in the identification of compounds with distinct mechanisms of action, for example by promoting host immune responses or by impeding fungal virulence factors. With the recent advent of using predictive models to screen for compounds with improved chances of bioavailability in the nematode a priori, high-throughput screening of chemical libraries using the C. elegans-C. albicans antifungal discovery assay holds even greater promise for the identification of novel antifungal agents in the near future.

Blocking Effect on Adsorption-Controlled Electrochemiluminescence of CdSe Nanoparticles for Detection of the Neurotransmitter Serotonin

Herein, we have designed a special strategy for detection of the important neurotransmitter serotonin by coupling the electrodeposition of serotonin oxidative products with the adsorption-controlled anodic electrochemiluminescent dynamics of the CdSe-triethylamine system. The stable electrochemiluminescent emission is found to decline in the presence of serotonin. This uncommon inhibition is attributed to the gradual accumulation of the electropolymerized serotonin products, which decrease the adsorbance of CdSe nanoparticles on the glass carbon electrode, as demonstrated by the scanning electron microscopic images (SEM) and the stripping voltammetric identification. Based on the inhibition effect, a facile strategy has been developed to sensitively detect the serotonin ranging from 0.2-10 μM, with a detection limit of 0.1 μM. The interference from the common species including ascorbic acid and uric acid has been investigated. The recovery rates in the spiked serum samples range from 94-112%, thereby implicating its potential applications in the complex bioassays.

Benefits and limits of sediment toxicity tests as an aid to decision‐making

With respect to the management of dredged sediments, a crucial issue is whether the removed materials (watered and/or processed) are disposed of or reused in an environmentally sound manner. In this context, the number of studies dealing with hazard or risk assessment has exponentially increased. This emphasis has resulted in the promotion and application of a very large variety of ecotoxicological tests. As a consequence, there is a clear need to highlight the scope and limitations of these tests for their appropriate selection and interpretation. In this paper we discuss the choice, implementation and interpretation of laboratory tests carried out on aquatic organisms at various levels of biological organization. We examine some experimental tools and methods in order to determine how suitable they are in regard to the objectives for which they are employed. To make this discussion more consistent our paper is based on results from research programmes conducted for governmental organizations and industrial partners. The Laboratoire des Sciences de l’Environnement (LSE) was involved with Cemagref de Lyon in a first methodological programme on risk assessment of scenarios of dredged sediment deposition. Another programme for a chemical company was implemented to assess the benefits of a physico‐chemical treatment applied to contaminated dredged sediments in a scenario of reuse or deposit in gravel pits. Currently, the LSE is working on a programme of risk assessment for road sediments in valorization scenarios. From these programmes, we expose how single‐species tests, as well as more complex bioassays and microcosm tests, can be used in an iterative step of risk assessment. Concerning microcosm tests, we also introduce a more realistic system that has been designed to simulate natural hydraulic conditions of gravel pits to assess the effects of toxicants on gravel pit aquatic biota during the sediment immersion phase and the sediment post‐deposition phase (paper in preparation). The benefits of these ecotoxicological approaches are underlined, but limits are discussed with regard to several criteria: ecological relevance, realism, use for decision‐making, cost and complexity of methods involved.

Yeast Barcoders: a chemogenomic application of a universal donor-strain collection carrying bar-code identifiers

The ability to perform complex bioassays in parallel enables experiments that are otherwise impossible because of throughput and cost constraints. For example, highly parallel chemical-genetic screens using pooled collections of thousands of defined Saccharomyces cerevisiae gene deletion strains are feasible because each strain is bar-coded with unique DNA sequences. It is, however, time-consuming and expensive to individually bar-code individual strains. To provide a simple and general method of barcoding yeast collections, we built a set of donor strains, called Barcoders, with unique bar codes that can be systematically transferred to any S. cerevisiae collection. We applied this technology by generating a collection of bar-coded 'decreased abundance by mRNA perturbation' (DAmP) loss-of-function strains comprising 87.1% of all essential yeast genes. These experiments validate both the Barcoders and the DAmP strain collection as useful tools for genome-wide chemical-genetic assays.

High-level synthesis of digital microfluidic biochips

Microfluidic biochips offer a promising platform for massively parallel DNA analysis, automated drug discovery, and real-time biomolecular recognition. Current techniques for full-custom design of droplet-based “digital” biochips do not scale well for concurrent assays and for next-generation system-on-chip (SOC) designs that are expected to include microfluidic components. We propose a system design methodology that attempts to apply classical high-level synthesis techniques to the design of digital microfluidic biochips. We focus here on the problem of scheduling bioassay functions under resource constraints. We first develop an optimal scheduling strategy based on integer linear programming. However, because the scheduling problem is NP-complete, we also develop two heuristic techniques that scale well for large problem instances. A clinical diagnostic procedure, namely multiplexed in-vitro diagnostics on human physiological fluids, is first used to illustrate and evaluate the proposed method. Next, the synthesis approach is applied to a protein assay, which serves as a more complex bioassay application. The proposed synthesis approach is expected to reduce human effort and design cycle time, and it will facilitate the integration of microfluidic components with microelectronic components in next-generation SOCs.

Fast, sensitive and selective liquid chromatographic–tandem mass spectrometric determination of tumor-promoting diterpene esters

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was developed to detect tumor-promoting diterpene esters of the tigliane and ingenane types within plant extracts. Fractionation on a C 18 high-performance liquid chromatography (HPLC) column was followed by MS–MS–multiple reaction monitoring (MRM) using the precursor→product ion pairs of m/z 311→293 and 293→265 for phorbol esters. The ion pairs m/z 313→295 and 295→267 were used for ingenol and deoxyphorbol esters. In a second run, the characteristic ions at m/z 311 and 313 were followed in precursor ion scan mode. These quasi-molecular ions were utilized to obtain full scan spectra of the compounds in product ion scan mode. Due to its selectivity, the present on-line method can be applied for plant cultivar selection and plant product control without time-consuming extraction procedures and complex bioassays.

Seminal Fluid Analysis and Sperm Function Testing

The diagnosis of male infertility is a rapidly developing field of investigation. The traditional descriptive approach to semen analysis is still at the heart of the diagnostic workup of male patients and important advances have been made in the standardization of the procedures used to construct the conventional semen profile, as embodied in the WHO handbook. Significant improvements have also been made in the techniques used to assess the quality of sperm motility. While this was once an entirely subjective exercise, the introduction of CASA systems to take objective measurements of the trajectories of human spermatozoa has revolutionized this form of analysis. The speed and accuracy of such systems has permitted detailed analyses of the relationships between sperm movement and sperm function, which has greatly enhanced the diagnostic power of this form of descriptive analysis. Notwithstanding the importance of the descriptive approach to semen analysis, it has also been recognized that to achieve an accurate diagnosis of male infertility such criteria should be supplemented with assays designed to reveal the functional competence of the spermatozoa. Bioassays have therefore been developed to measure such functions as the penetration of cervical mucus, sperm-zona interaction, the acrosome reaction, and sperm-oocyte fusion. All of these assays have been shown to generate information predictive of the fertilizing potential of human spermatozoa in vivo and in vitro. Despite their diagnostic value, the time, expense, and expertise required to run such functional assays has meant that they have not been widely used by infertility specialists. These functional assays are of value, however, in fundamental studies designed to elucidate the biochemical basis of defective sperm function. The first fruits of this research effort are now beginning to appear in the identification of a number of cytoplasmic markers for defective sperm function and the realization that lipid peroxidation plays a key role in the etiology of male infertility. In the wake of these fundamental studies will flow a new generation of biochemical tests for the diagnosis of defective sperm function and, it is hoped, the development of rational therapies with which to treat this condition.