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Regulating Biomolecular Surface Interactions Using Tunable Acoustic Streaming
Abstract
Diffusion limitations and nonspecific surface absorption are great challenges for developing micro-/nanoscale affinity biosensors. There are very limited approaches that can solve these issues at the same time. Here, an acoustic streaming approach enabled by a gigahertz (GHz) resonator is presented to promote mass transfer of analytes through the jet mode and biofouling removal through the shear mode, which can be switched by tuning the deviation angle, α, between the resonator and the sensor. Simulations show that the jet mode (α ≤ 0) drives the analytes in the fluid toward the sensing surface, overcomes the diffusion limitation, and enhances the binding; while the shear mode (0 < α < π/4) provides a scouring action to remove the biofouling from the sensor. Experimental studies were performed by integrating this GHz resonator with optoelectronic sensing systems, where a 34-fold enhancement for the initial binding rate was obtained. Featuring high efficiency, controllability, and versatility, we believe that this GHz acoustic streaming approach holds promise for many kinds of biosensing systems as well as lab-on-chip systems.
Point‐of‐care testing (POCT) has played important role in clinical diagnostics, environmental assessment, chemical and biological analyses, and food and chemical processing due to its faster turnaround compared to laboratory testing. Dedicated manipulations of solutions or particles are generally required to develop POCT technologies that achieve a “sample‐in‐answer‐out” operation. With the development of micro‐ and nanotechnology, many tools have been developed for sample preparation, on‐site analysis and solution manipulations (mixing, pumping, valving, etc.). Among these approaches, the use of acoustic waves to manipulate fluids and particles (named acoustofluidics) has been applied in many researches. This review focuses on the recent developments in acoustofluidics for POCT. It starts with the fundamentals of different acoustic manipulation techniques and then lists some of representative examples to highlight each method in practical POC applications. Looking toward the future, a compact, portable, highly integrated, low power, and biocompatible technique is anticipated to simultaneously achieve precise manipulation of small targets and multimodal manipulation in POC applications.
Contactless acoustic manipulation of micro/nanoscale particles has attracted considerable attention owing to its near independence of the physical and chemical properties of the targets, making it universally applicable to almost all biological systems. Thin-film bulk acoustic wave (BAW) resonators operating at gigahertz (GHz) frequencies have been demonstrated to generate localized high-speed microvortices through acoustic streaming effects. Benefitting from the strong drag forces of the high-speed vortices, BAW-enabled GHz acoustic streaming tweezers (AST) have been applied to the trapping and enrichment of particles ranging in size from micrometers to less than 100 nm. However, the behavior of particles in such 3D microvortex systems is still largely unknown. In this work, the particle behavior (trapping, enrichment, and separation) in GHz AST is studied by theoretical analyses, 3D simulations, and microparticle tracking experiments. It is found that the particle motion in the vortices is determined mainly by the balance between the acoustic streaming drag force and the acoustic radiation force. This work can provide basic design principles for AST-based lab-on-a-chip systems for a variety of applications.
Background
Controllable and multiple DNA release is critical in modern gene-based therapies. Current approaches require complex assistant molecules for combined release. To overcome the restrictions on the materials and environment, a novel and versatile DNA release method using a nano-electromechanical (NEMS) hypersonic resonator of gigahertz (GHz) frequency is developed.
Results
The micro-vortexes excited by ultra-high frequency acoustic wave can generate tunable shear stress at solid–liquid interface, thereby disrupting molecular interactions in immobilized multilayered polyelectrolyte thin films and releasing embedded DNA strands in a controlled fashion. Both finite element model analysis and experiment results verify the feasibility of this method. The release rate and released amount are confirmed to be well tuned. Owing to the different forces generated at different depth of the films, release of two types of DNA molecules with different velocities is achieved, which further explores its application in combined gene therapy.
Conclusions
Our research confirmed that this novel platform based on a nano-electromechanical hypersonic resonator works well for controllable single and multi-DNA release. In addition, the unique features of this resonator such as miniaturization and batch manufacturing open its possibility to be developed into a high-throughput, implantable and site targeting DNA release and delivery system.
Fluid flow and flow-induced shear stress are critical components of the vascular microenvironment commonly studied using microfluidic cell culture models. Microfluidic vascular models mimicking the physiological microenvironment also offer great potential for incorporating on-chip biomolecular detection. In spite of this potential, however, there are few examples of such functionality. Detection of biomolecules released by cells under flow-induced shear stress is a significant challenge due to severe sample dilution caused by the fluid flow used to generate the shear stress, frequently to the extent where the analyte is no longer detectable. In this work, we developed a computational model of a vascular microfluidic cell culture model that integrates physiological shear flow and on-chip monitoring of cell-secreted factors. Applicable to multilayer device configurations, the computational model was applied to a bilayer configuration, which has been used in numerous cell culture applications including vascular models. Guidelines were established that allow cells to be subjected to a wide range of physiological shear stress while ensuring optimal rapid transport of analyte to the biosensor surface and minimized biosensor response times. These guidelines therefore enable the development of microfluidic vascular models that integrate cell-secreted factor detection while addressing flow constraints imposed by physiological shear stress. Ultimately, this work will result in the addition of valuable functionality to microfluidic cell culture models that further fulfill their potential as labs-on-chips.
The inflammatory response in endothelial cells (ECs) leads to an increase in vascular permeability through the formation of gaps. However, the dynamic nature of vascular permeability and external factors involved is still elusive. In this work, we use a biomimetic blood vessel (BBV) microfluidic model to measure in real-time the change in permeability of the EC layer under culture in physiologically relevant flow conditions. This platform studies the dynamics and characterizes vascular permeability when the EC layer is triggered with an inflammatory agent using tracer molecules of three different sizes, and the results are compared to a transwell insert study. We also apply an analytical model to compare the permeability data from the different tracer molecules to understand the physiological and bio-transport significance of endothelial permeability based on the molecule of interest. A computational model of the BBV model is also built to understand the factors influencing transport of molecules of different sizes under flow. The endothelial monolayer cultured under flow in the BBV model was treated with thrombin, a serine protease that induces a rapid and reversible increase in endothelium permeability. On analysis of permeability data, it is found that the transport characteristics for fluorescein isothiocyanate (FITC) dye and FITC Dextran 4k Da molecules are similar in both BBV and transwell models, but FITC Dextran 70k Da molecules show increased permeability in the BBV model as convection flow (Peclet number?>?1) influences the molecule transport in the BBV model. We also calculated from permeability data the relative increase in intercellular gap area during thrombin treatment for ECs in the BBV and transwell insert models to be between 12% and 15%. This relative increase was found to be within range of what we quantified from F-actin stained EC layer images. The work highlights the importance of incorporating flow in in vitro vascular models, especially in studies involving transport of large size objects such as antibodies, proteins, nano/micro particles, and cells.
In clinical oncology, diagnosis and evaluation of optimal treatment strategies are mostly based on histopathological examination combined with immunohistochemical (IHC) expression analysis of cancer-associated antigens in formalin fixed paraffin-embedded (FFPE) tissue biopsies. However, informative IHC analysis depends on both the specificity and affinity of the binding reagent, which are inherently difficult to quantify in situ. Here we describe a label-free method that allows for the direct and real-time assessment of molecular binding kinetics in situ on FFPE tissue specimens using quartz crystal microbalance (QCM) enabled biosensor technology. We analysed the interaction between the rVAR2 protein and its placental-like chondroitin sulfate (pl-CS) receptor in primary human placenta tissue and in breast and prostate tumor specimens in situ. rVAR2 interacted with FFPE human placenta and cancer tissue with an affinity in the nanomolar range, and showed no detectable interaction with pl-CS negative normal tissue. We further validated the method by including analysis with the androgen receptor N-20 antibody (anti-AR). As the KD value produced by this method is independent of the number of epitopes available, this readout offers a quantitative and unbiased readout for in situ binding-avidity and amount of binding epitopes. In summary, this method adds a new and important dimension to classical IHC-based molecular pathology by adding information about the binding characteristics in biologically relevant conditions. This can potentially be used to select optimal biologics for diagnostic and for therapeutic applications as well as guide the development of novel high affinity binding drugs.
We report a novel optoelectrofluidic immunoreaction system based on electroosmotic flow for enhancing antibody–analyte binding efficiency on surface-based sensing system. Two conventional indium tin oxide glass slides are assembled to provide a reaction chamber for a tiny volume of sample droplet (~5 µL), in which the top layer is employed as an antibody-immobilized substrate and the bottom layer acts as a photoconductive layer of an optoelectrofluidic device. Under the application of AC voltage, an illuminated light pattern on the photoconductive layer causes strong counter-rotating vortices to transport analytes from bulk solution to the vicinity of the assay spot on the glass substrate. This configuration overcomes slow immunoreaction problem of diffusion-based sensing system, resulting in enhancement of binding efficiency via an optoelectrofulidic manner. Furthemore, we investigate the effect of optically-induced dynamic AC electroosmotic flow on optoelectrofluidic enhancement for surface-based immunoreaction with mathematical simulation study and real experiments using immunoglobulin G (IgG) and anti-IgG. As a result, dynamic light patterns provided better immunoreaction efficiency than static light patterns due to effective mass transport of target analyte, resulting in an achievement of 2.18-fold enhancement under a growing circular light pattern compared to the passive mode.
The demand for easy to use and cost effective medical technologies inspires scientists to develop innovative lab-on-chip technologies for point-of-care in vitro diagnostic testing. To fulfill medical needs, the tests should be rapid, sensitive, quantitative, and miniaturizable, and need to integrate all steps from sample-in to result-out. Here, we review the use of magnetic particles actuated by magnetic fields to perform the different process steps that are required for integrated lab-on-chip diagnostic assays. We discuss the use of magnetic particles to mix fluids, to capture specific analytes, to concentrate analytes, to transfer analytes from one solution to another, to label analytes, to perform stringency and washing steps, and to probe biophysical properties of the analytes, distinguishing methodologies with fluid flow and without fluid flow (stationary microfluidics). Our review focuses on efforts to combine and integrate different magnetically actuated assay steps, with the vision that it will become possible in the future to realize integrated lab-on-chip biosensing assays in which all assay process steps are controlled and optimized by magnetic forces.
Non-invasive real-time observations and the evaluation of living cell conditions and functions are increasingly demanded in life sciences. Surface plasmon resonance (SPR) sensors detect the refractive index (RI) changes on the surface of sensor chips in label-free and on a real-time basis. Using SPR sensors, we and other groups have developed techniques to evaluate living cells' reactions in response to stimuli without any labeling in a real-time manner. The SPR imaging (SPRI) system for living cells may visualize single cell reactions and has the potential to expand application of SPR cell sensing for clinical diagnosis, such as multi-array cell diagnostic systems and detection of malignant cells among normal cells in combination with rapid cell isolation techniques.
Field-effect transistor-based biosensors (bioFETs) have shown great promise in the field of fast, ultra-sensitive, label-free detection of biomolecules. Reliability and accuracy, when trying to measure small concentrations, is of paramount importance for the translation of these research devices into the clinical setting. Our knowledge and experience with these sensors has reached a stage where we are able to identify three main aspects of bioFET sensing that currently limit their applications. By considering the intrinsic device noise as a limitation to the smallest measurable signal, we show how various parameters, processing steps and surface modifications, affect the limit of detection. We also introduce the signal-to-noise ratio of bioFETs as a universal performance metric, which allows us to gain better insight into the design of more sensitive devices. Another aspect that places a limit on the performance of bioFETs is screening by the electrolyte environment, which reduces the signal that could be potentially measured. Alternative functionalization and detection schemes that could enable the use of these charge-based sensors in physiological conditions are highlighted. Finally, the binding kinetics of the receptor–analyte system are considered, both in the context of extracting information about molecular interactions using the bioFET sensor platform and as a fundamental limitation to the number of molecules that bind to the sensor surface at steady-state conditions and to the signal that is generated. Some strategies to overcome these limitations are also proposed. Taken together, these performance-limiting issues, if solved, would bring bioFET sensors closer to clinical applications. WIREs Nanomed Nanobiotechnol 2013, 5:629–645. doi: 10.1002/wnan.1227
Conflict of interest: The authors have declared no conflicts of interest for this article.
For further resources related to this article, please visit the WIREs website.
Affinity-based biosensing systems have become an important analytical tool for the detection and study of numerous biomolecules. The merging of these sensing technologies with microfluidic flow cells allows for faster detection times, increased sensitivities, and lower required sample volumes. In order to obtain a higher degree of performance from the sensor, it is important to know the effects of the flow cell geometry on the sensor sensitivity. In these sensors, the sensor sensitivity is related to the overall diffusive flux of analyte to the sensing surface; therefore increases in the analyte flux will be manifested as an increase in sensitivity, resulting in a lower limit of detection (LOD). Here we present a study pertaining to the effects of the flow cell height H on the analyte flux J, where for a common biosensor design we predict that the analyte flux will scale as . We verify this scaling behavior via both numerical simulations as well as an experimental surface plasmon resonance (SPR) biosensor. We show the reduction of the flow cell height can have drastic effects on the sensor performance, where the LOD of our experimental system concerning the detection of ssDNA decreases by a factor of 4 when H is reduced from 47 μm to 7 μm. We utilize these results to discuss the applicability of this scaling behavior with respect to a generalized affinity-based biosensor.
Monitoring the binding affinities and kinetics of protein interactions is important in clinical diagnostics and drug development because such information is used to identify new therapeutic candidates. Surface plasmon resonance is at present the standard method used for such analysis, but this is limited by low sensitivity and low-throughput analysis. Here, we show that silicon nanowire field-effect transistors can be used as biosensors to measure protein-ligand binding affinities and kinetics with sensitivities down to femtomolar concentrations. Based on this sensing mechanism, we develop an analytical model to calibrate the sensor response and quantify the molecular binding affinities of two representative protein-ligand binding pairs. The rate constant of the association and dissociation of the protein-ligand pair is determined by monitoring the reaction kinetics, demonstrating that silicon nanowire field-effect transistors can be readily used as high-throughput biosensors to quantify protein interactions.
We demonstrate a technique to recirculate liquids in a microfluidic channel by alternating predominance of centrifugal and capillary forces to rapidly bring the entire volume of a liquid sample to within one diffusion length, delta, of the surface, even for sample volumes hundreds of times the product of delta and the geometric device area. This is accomplished by repetitive, random sampling of an on-disc sample reservoir to form a thin fluid layer of thickness delta in a microchannel, maintaining contact for the diffusion time, then rapidly exchanging the fluid layer for a fresh aliquot by disc rotation and stoppage. With this technique, liquid volumes of microlitres to millilitres can be handled in many sizes of microfluidic channels, provided the channel wall with greatest surface area is hydrophilic. We present a theoretical model describing the balance of centrifugal and capillary forces in the device and validate the model experimentally.
Mechanical interactions are fundamental to biology. Mechanical forces of chemical origin determine motility and adhesion on the cellular scale, and govern transport and affinity on the molecular scale. Biological sensing in the mechanical domain provides unique opportunities to measure forces, displacements and mass changes from cellular and subcellular processes. Nanomechanical systems are particularly well matched in size with molecular interactions, and provide a basis for biological probes with single-molecule sensitivity. Here we review micro- and nanoscale biosensors, with a particular focus on fast mechanical biosensing in fluid by mass- and force-based methods, and the challenges presented by non-specific interactions. We explain the general issues that will be critical to the success of any type of next-generation mechanical biosensor, such as the need to improve intrinsic device performance, fabrication reproducibility and system integration. We also discuss the need for a greater understanding of analyte-sensor interactions on the nanoscale and of stochastic processes in the sensing environment.
A droplet-based micro-total-analysis system involving biosensor performance enhancement by integrated surface-acoustic-wave (SAW) microstreaming is shown. The bioreactor consists of an encapsulated droplet with a biosensor on its periphery, with in situ streaming induced by SAW. This paper highlights the characterization by particle image tracking of the speed distribution inside the droplet. The analyte-biosensor interaction is then evaluated by finite element simulation with different streaming conditions. Calculation of the biosensing enhancement shows an optimum in the biosensor response. These results confirm that the evaluation of the Damköhler and Peclet numbers is of primary importance when designing biosensors enhanced by streaming.
This article presents a device incorporating surface plasmon resonance (SPR) sensing and surface acoustic wave (SAW) actuation integrated onto a common LiNbO(3) piezoelectric substrate. The device uses Rayleigh-type SAW to provide active microfluidic mixing in the fluid above the SPR sensor. Validation experiments show that SAW-induced microfluidic mixing results in accelerated binding kinetics of an avidin-biotin assay. Results also show that, though SAW action causes a parasitic SPR response due to heat injection into the fluid, a relatively brief relaxation time following the SAW pulses allows the effect to dissipate, without affecting the overall assay response. Since both SPR sensors and SAW transducers can be fabricated simultaneously using low-cost microfabrication methods on a single substrate, the proposed design is well-suited to lab-on-chip applications.
We describe highly sensitive, label-free, multiplexed electrical detection of cancer markers using silicon-nanowire field-effect devices in which distinct nanowires and surface receptors are incorporated into arrays. Protein markers were routinely detected at femtomolar concentrations with high selectivity, and simultaneous incorporation of control nanowires enabled discrimination against false positives. Nanowire arrays allowed highly selective and sensitive multiplexed detection of prostate specific antigen (PSA), PSA-alpha1-antichymotrypsin, carcinoembryonic antigen and mucin-1, including detection to at least 0.9 pg/ml in undiluted serum samples. In addition, nucleic acid receptors enabled real-time assays of the binding, activity and small-molecule inhibition of telomerase using unamplified extracts from as few as ten tumor cells. The capability for multiplexed real-time monitoring of protein markers and telomerase activity with high sensitivity and selectivity in clinically relevant samples opens up substantial possibilities for diagnosis and treatment of cancer and other complex diseases.
AC-driven electrothermal flow is used to enhance the temporal performance of heterogeneous immuno-sensors in microfluidic systems by nearly an order of magnitude. AC electrokinetic forces are used to generate electrothermal flow, which in turn produces a circular stirring fluid motion that enhances the transport of diffusion-limited proteins. This provides more binding opportunities between suspended antigens and wall-immobilized antibodies. We investigate experimentally the effectiveness of electrothermal stirring, using a biotin-streptavidin heterogeneous assay, in which biotin is immobilized, and fluorescently-labeled streptavidin is suspended in a high conductivity buffer (sigma = 1.0 S m(-1)). Microfabricated electrodes were integrated within a microwell and driven at a frequency of f= 200 kHz and 10 V(rms). Fluorescent intensity measurements show that for a five minute assay, electrothermal stirring increases the binding rate by a factor of almost nine. Similar binding improvement was measured for longer assays, up to fifteen minutes. The electrothermal enhancement of this assay was modeled numerically and agrees with experimental binding rates. The measured fluid velocity of 22 +/- 2 microm s(-1) was significantly lower than that predicted by the numerical model, 1.1 mm s(-1), but nevertheless shows the same fourth power dependence on applied potential. The results demonstrate the ability for electrothermal stirring to reliably improve the response time and sensitivity within a given time limit for microfluidic diffusion-limited sensors.
The past decade has seen researchers develop and apply novel technologies for biomolecular detection, at times approaching hard limits imposed by physics and chemistry. In nearly all sensors, the transport of target molecules to the sensor can play as critical a role as the chemical reaction itself in governing binding kinetics, and ultimately performance. Yet rarely does an analysis of the interplay between diffusion, convection and reaction motivate experimental design or interpretation. Here we develop a physically intuitive and practical understanding of analyte transport for researchers who develop and employ biosensors based on surface capture. We explore the qualitatively distinct behaviors that result, develop rules of thumb to quickly determine how a given system will behave, and derive order-of-magnitude estimates for fundamental quantities of interest, such as fluxes, collection rates and equilibration times. We pay particular attention to collection limits for micro- and nanoscale sensors, and highlight unexplained discrepancies between reported values and theoretical limits.
Nonspecific binding is a universal problem that reduces bioassay sensitivity and specificity. We demonstrate that ultrasonic waves, generated by 5-MHz quartz crystal resonators, accelerate nonspecifically bound protein desorption from sensing and nonsensing areas of micropatterned protein arrays, controllably and nondestructively cleaning the micropatterns. Nonsensing area fluorescent intensity values dropped by more than 85% and sensing area fluorescent intensity dropped 77% due to nonspecific binding removal at an input power of 14 W. After patterning, antibody films were many layers thick with nonspecifically bound protein, and aggregates obscured patterns. Quartz crystal resonators removed excess antibody layers and aggregates leaving highly uniform films, as evidenced by smaller spatial variations in fluorescent intensity and atomic force microscope surface roughness values. Fluorescent intensity values obtained after 14-W QCR operation were more repeatable and uniform.
We present a method for monitoring spatially localized antigen-antibody binding events on physiologically relevant substrates (cell and tissue sections) using fluorescence lifetime imaging. Specifically, we use the difference between the fluorescence decay times of fluorescently tagged antibodies in free solution and in the bound state to track the bound fraction over time and hence deduce the binding kinetics. We make use of a microfluidic probe format to minimize the mass transport effects and localize the analysis to specific regions of interest on the biological substrates. This enables measurement of binding constants (kon) on surface-bound antigens and on cell blocks using model biomarkers. Finally, we directly measure p53 kinetics with differential biomarker expression in ovarian cancer tissue sections, observing that the degree of expression corresponds to the changes in kon, with values of 3.27-3.50 × 103 M-1 s-1 for high biomarker expression and 2.27-2.79 × 103 M-1 s-1 for low biomarker expression.
Surface assays, such as ELISA and immunofluorescence, are nothing short of ubiquitous in biotechnology and medical diagnostics today. The development and optimization of these assays generally focuses on three aspects: immobilization chemistry, ligand-receptor interaction and concentrations of ligands, buffers and sample. A fourth aspect, the transport of the analyte to the surface, is more rarely delved into during assay design and analysis. Improving transport is generally limited to the agitation of reagents, a mode of flow generation inherently difficult to control, often resulting in inconsistent reaction kinetics. However, with assay optimization reaching theoretical limits, the role of transport becomes decisive. This perspective develops an intuitive and practical understanding of transport in conventional agitation systems and in microfluidics, the latter underpinning many new life science technologies. We give rules of thumb to guide the user on system behavior, such as advection regimes and shear stress, and derive estimates for relevant quantities that delimit assay parameters. Illustrative cases with examples of experimental results are used to clarify the role of fundamental concepts such as boundary and depletion layers, mass diffusivity or surface tension.
Biosensing based on optical micro- and nano-resonators integrated in a microfluidics environment is a promising approach to lab-on-a-chip platforms capable of detecting low concentrations of analytes from small sample volumes. While sensitivity has reached the single molecule level, in practice, the applicability to real-life settings is limited by Brownian diffusion of the analyte to the sensor surface, which dictates the total duration of the sensing assay. Here, we use the electrothermoplasmonic (ETP) effect to overcome this limit through opto-electrical fluid convective flow generation. To this end, we designed a Localized Surface Plasmon Resonance (LSPR) sensing chip that integrates ETP operation into state-of-the-art microfluidics. First, we optimize and characterize the ETP dynamics inside the microfluidic chamber, showing high fluid velocities. Then, we perform proof-of-concept experiments on model immunoglobulin G detection to demonstrate ETP-enhanced biosensing. Our results demonstrate the synergetic effect of temperature and electric field proving that ETP-LSPR has improved performances over standard LSPR.
We report the nonlinear acoustic streaming effect and the fast manipulation of microparticles by microelectromechanical Lamb-wave resonators in a microliter droplet. The device, consisting of four Lamb-wave resonators on a silicon die, generates cylindrical traveling waves in a liquid and efficiently drives nine horizontal vortices within a 1−μl droplet; the performance of the device coincides with the numerical model prediction. Experimentally, the particles are enriched at the stagnation center of the main vortex on the free surface of the droplet in open space without microfluidic channels. In addition, the trajectories of the particles in the droplet can be controlled by the excitation power.
Nonspecific binding (NSB) is a general issue for surface based biosensors. Various approaches have been developed to prevent or remove the NSBs. However, these approaches either increased the background signals of the sensors or limited to specific transducers interface. In this work, we developed a hydrodynamic approach to selectively remove the NSBs using a microfabricated hypersonic resonator with 2.5 gigahertz (GHz) resonant frequency. The high frequency device facilitates to generate multiple controlled micro-vortices which then create cleaning forces at the solid-liquid interfaces. The competitive adhesive and cleaning forces have been investigated using the finite element method (FEM) simulation, identifying the feasibility of the vortices induced NSB removal. NSB proteins have been selectively removed experimentally both on the surface of the resonator and on other substrates which contact the vortices. Thus, the developed hydrodynamic approach is believed to be a simple and versatile tool for NSB removal and compatible to many sensor systems. The unique feature of the hypersonic resonator is that it can be used as a gravimetric sensor as well, thus a combined NSB removal and protein detection dual functional biosensor system is developed.
Fibrin is a plasma protein with a central role in blood clotting and wound repair. Upon vascular injury, fibrin forms resilient fibrillar networks (clots) via a multi-step self-assembly process, from monomers, to double-stranded protofibrils, to a branched network of thick fibers. In vitro, fibrin self-assembly is sensitive to physicochemical conditions like the solution pH and ionic strength, which tune the strength of the non-covalent driving forces. Here we report a surprising finding that the buffer—which is necessary to control the pH and is typically considered to be inert—also significantly influences fibrin self-assembly. We show by confocal microscopy and quantitative light-scattering that various common buffering agents have no effect on the initial assembly of fibrin monomers into protofibrils but strongly hamper the subsequent lateral association of protofibrils into thicker fibers. We further find that the structural changes are independent of the molecular structure of the buffering agents as well as of the activation mechanism, and even occur in fibrin networks formed from platelet-poor plasma. This buffer-mediated decrease in protofibril bundling results in a marked reduction in the permeability of fibrin networks, but only weakly influences the elastic modulus of fibrin networks, providing a useful tuning parameter to independently control the elastic properties and the permeability of fibrin networks. Our work raises the possibility that fibrin assembly in vivo may be regulated by variations in the acute-phase levels of bicarbonate and phosphate, which act as physiological buffering agents of blood pH. Moreover, our findings add a new example of buffer-induced effects on biomolecular self-assembly to recent findings for a range of proteins and lipids.
In this work, gigahertz solidly mounted resonators (SMRs) (2.5 GHz) were designed and fabricated to construct a novel particle-resonator system to achieve the biomolecular stiffness sensing in real time. The positive frequency shift of the system was used to estimate the stiffness of biomolecules connecting between the SMR and attached particles. The working principle was revealed by the mathematical analysis of the general block-spring model of the system. Further interpretations about the mechanism of such elastic interaction from the perspective of acoustic resonant modes of SMRs were given by finite element method. Biotin-streptavidin, antibody and antigen binding system were used as model molecular linkers to study the frequency shift varied with different particle diameters and particle densities. Different linker stiffness was realized by adjusting the concentrations of antigens connected with particles which form specific binding with antibodies immobilized on the SMR. The results fairly agree with the simulation results demonstrating the proposed particle-resonator system as an effective method to realize the real-time biomolecular stiffness detection.
Label-free, nanoparticle-based plasmonic optical biosensing, combined with device miniaturization and microarray integration, has emerged as a promising approach for rapid, multiplexed biomolecular analysis. However, limited sensitivity prevents the wide use of such integrated label-free nanoplasmonic biosensors in clinical and life science applications where low-abundance biomolecule detection is needed. Here, we present a nano-plasmofluidic device integrated with microelectrodes for rapid, label-free analysis of a low-abundance cell signaling protein, detected by AC electroosmosis-enhanced localized surface plasmon resonance (ACE-LSPR) biofunctional nanoparticle imaging. The ACE-LSPR device is constructed using both bottom-up and top-down sensor fabrication methods, allowing the seamless integration of antibody-conjugated gold nanorod (AuNR) biosensor arrays with microelectrodes on the same microfluidic platform. Applying an AC voltage to microelectrodes while scanning the scattering light intensity variation of the AuNR biosensors results in significantly enhanced biosensing performance. The AC electroosmosis (ACEO) based enhancement of the biosensor performance enables rapid (5-15 min) quantification of IL-1β, a pro-inflammatory cytokine biomarker, with a sensitivity down to 158.5 fg/mL (9.1 fM) for spiked samples in PBS and 1 pg/mL (58 fM) for diluted human serum. Together with the optimized detection sensitivity and speed, our study presents the first critical step towards the application of nanoplasmonic biosensing technology to immune status monitoring guided by low-abundance cytokine measurement.
This paper reports a surface functionalization strategy for protein detections based on biotin-derivatized poly(L-lysine)-grafted oligo-ethylene glycol (PLL-g-OEGx-Biotin) copolymers. Such strategy can be used to attach the biomolecule receptors in a reproducible way simply by incubation of the transducer element in a solution containing such copolymers which largely facilitated the sensor functionalization at an industrial scale. As the synthesized copolymers are cationic in physiology pH, surface biotinylation can be easily achieved via electrostatic adsorption on negatively charged sensor surface. Biotinylated receptors can be subsequently attached through well-defined biotin-streptavidin interaction. In this work, the bioactive sensor surfaces were applied for mouse IgG and prostate specific antigen (PSA) detections using quartz crystal microbalance (QCM), optical sensor (BioLayer Interferometry) and conventional ELISA test (colorimetry). A limit of detection (LOD) of 0.5 nM was achieved for PSA detections both in HEPES buffer and serum dilutions in ELISA tests. The synthesized PLL-g-OEGx-Biotin copolymers with different OEG chain length were also compared for their biosensing performance. Moreover, the surface regeneration was achieved by pH stimulation to remove the copolymers and the bonded analytes, while maintaining the sensor reusability as well. Thus, the developed PLL-g-OEGx-Biotin surface assembling strategy is believed to be a versatile surface coating method for protein detections with multi-sensor compatibility.
Zwitterionic peptides were anchored to a conducting polymer of citrate doped poly(3,4-ethylenedioxythiophene) (PEDOT) via the nickel cation coordination, and the obtained peptide modified PEDOT, with excellent antifouling ability and good conductivity, was further used for the immobilization of a DNA probe to construct an electrochemical biosensor for the breast cancer marker BRCA1. The DNA biosensor was highly sensitive (with detection limit of 0.03 fM) and selective, and it was able to detect BRCA1 in 5% (V/V) human plasma with satisfying accuracy and low fouling. The marriage of antifouling and biocompatible peptides with conducting polymers opened a new avenue to construct electrochemical biosensors capable of assaying targets in complex biological media with high sensitivity and without biofouling.
This investigation is devoted to the influences of non-uniform wall characteristics on the surface adsorption-desorption rates in an electrokinetic microarray. Utilizing already explored electroosmotic and electrophoretic velocities, the species transport equations are solved by a finite-volume-based numerical approach. Uniform, sinusoidal, and pulse-like distributions of the zeta potential are considered in the analysis. The developed model is validated by comparing the results with those of two analytical solutions that are derived for limiting conditions. The results reveal that, in some cases, the surface charge heterogeneity can reduce the saturation time by more than 60%. The efficacy of the surface charge heterogeneity is limited to high Damkohler numbers and its effects are negligible for small values of this parameter. Whereas the impact of non-uniform surface characteristics is amplified by increasing the Peclet number, it is reduced by increasing either of the channel length to radius ratio or the electrophoretic mobility of the analytes.
Enhancing the time response and detection limit of affinity-binding based biosensors is an area of active research. For diffusion limited reactions, introducing active mass transport is an effective strategy to reduce the equilibration time and improve surface binding. Here, a laser is focused on the ceiling of a microchamber to generate point heating, which introduces natural advection and thermophoresis to promote mass transport to the reactive floor. We first used COMSOL simulation to study how the kinetics of ligand binding is influenced by the optothermal effect. Afterwards, binding of biotinylated nanoparticles to NeutrAvidin-treated substrates are quantitatively measured with and without laser heating. It is discovered that laser induced point heating reduces reaction half-life locally, and the reduction improves with the natural advection velocity. In addition, non-uniform ligand binding on the substrate is induced by the laser with predicatable binding patterns. This optothermal strategy holds a promise to improve time-response and sensitivity of biosensor and microarrays.
In this study we examine the use of the staggered herringbone mixer (SHM) to increase the efficiency of analyte delivery to a planar biosensor surface. Although there has been an extensive amount of research regarding the optimization of the SHM for mixing purposes, there has been very little work regarding the use of said micromixers for sensing purposes. Here, we use numerical methods to examine the effect of the SHM geometry on the efficiency of analyte delivery to the sensor surface. We further show the level of sensing enhancement of an SHM-based sensing chamber over that of an unmixed chamber has a strong dependence on the SHM geometry, the Péclet number, and the overall sensor length. The results presented herein are applicable to a very wide range of biosensor transduction mechanisms and target analytes.
We report a simple method to remove nonspecifically adsorbed species from sensor surface and also improve the detection sensitivity of the sensor using tuneable alternating current (ac) electrohydrodynamics (ac-EHD) forces. These forces generated within few nanometers of an electrode surface (i.e., double layer) engender fluid flow within a serpentine channel containing a long array of the asymmetric electrode pairs, and can easily be tuned externally by changing the frequency and amplitude of the ac-EHD field. Under the optimized experimental conditions, we achieved a 3.5-fold reduction in nonspecific adsorption of non-target proteins with a 1000-fold enhancement in detection sensitivity of the device for the analysis of human epidermal growth factor receptor 2 (HER2) protein spiked in serum. This approach can be applicable in diverse fields including biosensors, cellular and molecular separation systems and biomedical applications to remove/reduce nonspecific adsorption of molecular and cellular species.
We outline a comprehensive numerical procedure for modeling of species transport and surface reaction kinetics in electrokinetically actuated microfluidic devices of rectangular cross section. Our results confirm the findings of previous simplified approaches that a concentration wave is created for sufficiently long microreactors. An analytical solution, developed for the wave propagation speed, shows that, when normalizing with the fluid mean velocity, it becomes a function of three parameters comprising the channel aspect ratio, the relative adsorption capacity, and the kinetic equilibrium constant. Our studies also reveal that the reactor geometry idealized as a slit, instead of a rectangular shape, gives rise to the underestimation of the saturation time. The extent of this underestimation increases by increasing the Damkohler number or decreasing the dimensionless Debye-Hückel parameter. Moreover, increasing the values of the Damkohler number, the dimensionless Debye-Hückel parameter, the relative adsorption capacity, and the velocity scale ratio results in lower saturation times.
This paper addresses the problem of detecting minute concentrations (nano to pico-molar) of analyte in a fluid flow chamber using an array of surface-based sensors. It is shown that in the mass-transport influenced case, when the rate of transport of analyte is comparable to or smaller than the intrinsic reaction rates at the sensor surface, substantial improvements in the response rate can be obtained from an array of spaced small sensor surfaces relative to a single large surface. Advection-diffusion-reaction models are developed to predict the response of such sensor arrays, which are compared to individual sensor surfaces of the same total area. Formulas are derived for quantifying the improvement in performance and optimal size of the sensors in the array. The results of the model are compared with experimental data obtained for an ion-channel switch biosensor and a surface plasmon resonance biosensor.
A design incorporating surface plasmon resonance (SPR) biosensing and surface acoustic wave (SAW) active microfluidic mixing, integrated on a single LiNbO3 piezoelectric substrate, is presented. Validation experiments show that SAW-mixing (microstreaming) results in accelerated binding kinetics (time-to-saturation) for a standard assay with appropriate SAW excitation parameters. Since both SPR sensors and SAW transducers can be fabricated simultaneously using low-cost microfabrication methods, the proposed design should contribute to improved lab-on-chip devices for detecting and identifying biomolecules of interest with greater accuracy and speed across multiple applications.
AC electroosmosis (ACEO) flow and label-free electrochemical impedance spectroscopy are employed to increase the hybridization rate and specifically detect target DNA (tDNA) concentrations. A low-ionic-strength solution, 6.1μS/cm 1mM Tris (pH 9.3), was used to produce ACEO and proved the feasibility of hybridization. Adequate voltage parameters for the simultaneous ACEO driving and DNA hybridization in the 1mM Tris solution were 1.5 V(pp) and 200Hz. Moreover, an electrode set with a 1:4 ring width-to-disk diameter ratio exhibited a larger ACEO velocity above the disk electrode surface to improve collecting efficiency. The ACEO-integrated DNA sensing chips could reach 90% saturation hybridization within 117s. The linear range and detection limit of the sensors was 10aM-10pM and 10aM, respectively. The label-free impedimetric DNA sensing chips with integrated ACEO stirring can perform rapid hybridization and highly-sensitive detections to specifically measure tDNA concentrations.
Surface plasmon resonance (SPR) is now widely embraced as a technology for monitoring a diverse range of protein-protein interactions and is considered almost de rigueur for characterizing antibody-antigen interactions. The technique obviates the need to label either of the interacting species and the binding event is visualized in real-time. Thus, it is ideally suited for screening crude, unpurified antibody samples that dominate early candidate panels following antibody selection campaigns. SPR returns both concentration and affinity data but when used correctly can also resolve the discrete component kinetic parameters (association and dissociation rate constants) of the affinity interaction. Herein, we outline some SPR-based generic antibody screening configurations and methodologies in the context of expediting data-rich ranking of candidate antibody panels and ensuring that antibodies with the optimal kinetic binding characteristics are reliably identified.
We present a disposable microarray hybridization chamber with an integrated micropump to speed up diffusion based reaction kinetics by generating convective flow. The time-to-result for the hybridization reaction was reduced from 60 min (standard protocol) down to 15 min for a commercially available microarray. The integrated displacement micropump is pneumatically actuated. It includes two active microvalves and is designed for low-cost, high volume manufacturing. The setup is made out of two microstructured polymer parts realized in polycarbonate (PC) separated by a 25 μm thermoplastic elastomer (TPE) membrane. Pump rate can be controlled between 0.3 μl s(-1) and 5.7 μl s(-1) at actuation frequencies between 0.2 Hz and 8.0 Hz, respectively.
Lectins are the proteins which can distinguish glycosylation patterns. They are frequently used as biomarkers for progressions of several diseases including cancer. As the lectin microarray based prognosis devices miniaturize the process of glycoprofiling, it is anticipated that their performance can be augmented by integration with microfluidic framework. This is analogous to microfluidics based DNA arrays. However, unlike small oligonucleotide microarrays, it remains uncertain whether the binding reaction-kinetic parameters can be considered invariant of imposed hydrodynamics, for relatively larger and structure sensitive molecules such as lectins. Here we show, using two standard lectins namely Concanavalin A and Abrus Agglutinin, that the steady state binding efficiency unexpectedly declines beyond a critical shear rate magnitude. This observation can be explained only if the associated reaction constants are presumed to be functions of hydrodynamic parameters. We methodically deduce the shear rate dependence of association and dissociation constants from the comparison of experimental and model-simulation trends. The aforementioned phenomena are perceived to be the consequences of strong hydrodynamic perturbations, culminating into molecular structural distortion. The exploration, therefore, reveals a unique coupling between reaction kinetics and hydrodynamics for biomacromolecules and provides a generic scheme towards futuristic microfluidics-coupled biomedical assays.
Human embryonic stem cell (hESC) derived cardiomyocytes are in the present study being used for testing drug-induced cardiotoxicity in a biosensor set-up. The design of an in vitro testing alternative provides a novel opportunity to surpass previous methods based on rodent cells or cell lines due to its significantly higher toxicological relevance. In this report we demonstrate how hESC-derived cardiomyocytes release detectable levels of two clinically decisive cardiac biomarkers, cardiac troponin T and fatty acid binding protein 3, when the cardiac cells are exposed to the well-known cardioactive drug compound, doxorubicin. The release is monitored by the immuno-biosensor technique surface plasmon resonance, particularly appropriate due to its capacity for parallel and high-throughput analysis in complex media.
The efficiency of a pre-absorbed bovine serum albumin (BSA) layer in blocking the non-specific adsorption of different proteins on hydrophobic and hydrophilic surfaces was evaluated qualitatively and quantitatively using infrared reflection spectroscopy supported by spectral simulations. A BSA layer with a surface coverage of 35% of a close-packed monolayer exhibited a blocking efficiency of 90-100% on a hydrophobic and 68-100% on a hydrophilic surface, with respect to the non-specific adsorption of concanavalin A (Con A), immunoglobulin G (IgG), and staphylococcal protein A (SpA). This BSA layer was produced using a solution concentration of 1 mg/mL and 30 min incubation time. BSA layers that were adsorbed at conditions commonly employed for blocking (a 12 h incubation time and a solution concentration of 10 mg/mL) exhibited a blocking activity that involved competitive adsorption-desorption. This activity resulted from the formation of BSA-phosphate surface complexes, which correlated with the conformation of adsorbed BSA molecules that was favourable for blocking. The importance of optimisation of the adsorbed BSA layer for different surfaces and proteins to achieve efficient blocking was addressed in this study.
A sandwich mixer consists of mixing two solutions in a channel, one central laminar flow being sandwiched between two outer flow solutions. The present numerical study considers the convection-diffusion of two reacting species A and B, provided respectively by the two incoming solutions. The simulations show how the diffusion coefficient, flow rate and species concentration ratios influence, via the transversal diffusion length and reaction kinetics, the reaction extent at the end of the sandwich mixer. First, this extent can be enhanced up to 60% if the species with the lowest diffusion coefficient is located in the outer solutions where the flow velocity is small compared to that of the central part (higher residence time). Secondly, decreasing the outer flow rates (to confine the reaction close to the walls) and increasing the local concentration to keep the same flux ratio improve the extent by 300%. Comparison with a bi-lamination passive mixer, with an ideal mixer and an electro-osmotic driven flow mixer is presented. These conclusions are also demonstrated for consecutive reactions, showing an amplification of the effects described above. The results are also presented versus the residence time in the mixer-reactor to show the time window for which the gain is appreciable.
We examine through analytical calculations and finite element simulations how the detection efficiency of disk and wire-like biosensors in unmixed fluids varies with size from the micrometer to nanometer scales. Specifically, we determine the total flux of DNA-like analyte molecules on a sensor as a function of time and flow rate for a sensor incorporated into a microfluidic system. In all cases, sensor size and shape profoundly affect the total analyte flux. The calculations reveal that reported femtomolar detection limits for biomolecular assays are very likely an analyte transport limitation, not a signal transduction limitation. We conclude that without directed transport of biomolecules, individual nanoscale sensors will be limited to picomolar-order sensitivity for practical time scales.
A novel anti-fouling mechanism based on the combined effects of electric field and shear stress is reported. A lead zirconate titanate (PZT) composite is used to generate an electric field and an acoustic streaming shear stress that increase nanomolecule desorption. In vitro characterization showed that (1) 58+/-5.5% and 39+/-5.2% of adsorbed bovine serum albumin (BSA) proteins can be effectively removed from fired silver and titanium coated PZT plate, respectively; and (2) 43+/-9.7% of the anti-mouse immunoglobulin G (IgG) can be effectively removed from a fired silver coated PZT plate. Theoretical calculations on protein-surface interactions (van der Waals (VDW), electrostatic, and hydrophobic) and shear stress describe the mechanism for protein desorption from model surfaces. We have shown that the applied electric potential is the major contributor in reducing the adhesive force between protein and surface, and the desorbed protein is taken away by acoustic streaming shear stress. We strongly believe that the present method offers the possibility of minimizing nanomolecule adsorption without further surface treatment.
This study examined six different polymer and self-assembled monolayer (SAM) surface modifications for their interactions with human serum and plasma. It was demonstrated that zwitterionic polymer surfaces are viable alternatives to more traditional surfaces based on poly(ethylene glycol) (PEG) as nonfouling surfaces. All polymer surfaces were formed using atom transfer radical polymerization (ATRP) and they showed an increased resistance to nonspecific protein adsorption compared to SAMs. This improvement is due to an increase in the surface packing density of nonfouling groups on the surface, as well as a steric repulsion from the flexible polymer brush surfaces. The zwitterionic polymer surface based on carboxybetaine methacrylate (CBMA) also incorporates functional groups for protein immobilization in the nonfouling background, making it a strong candidate for many applications such as in diagnostics and drug delivery.
Nonspecific binding of proteins is an ongoing problem that dramatically reduces the sensitivity and selectivity of biosensors. We demonstrate that ultrasonic waves generated by surface acoustic wave (SAW) devices remove nonspecifically bound proteins from the sensing and nonsensing regions of the microarrays. We demonstrate our approach for controllably and nondestructively cleaning the microarray interface. In this work, SAWs were generated using 128 YX lithium niobate, chosen for its high coupling coefficient and efficient power transfer to mechanical motion. These waves propagating along the surface were coupled into specifically bound and nonspecifically bound proteins on a patterned surface of 40 mum feature size. Fluorescence intensity was used to quantify cleaning efficacy of the microarrays. Our results have shown that excess protein layers and aggregates are removed leaving highly uniform films as evidenced by fluorescence intensity profiles. Selected antigen-receptor interactions remained bound during the acoustic cleaning process when subjected to 11.25 mW of power and retained their efficacy for subsequent antigen capture. Results demonstrate near-complete fluorescence signal recovery for both the sensing and nonsensing regions of the microarrays. Of significance is that our approach can be integrated into existing array technologies where sensing and nonsensing regions are extensively fouled. We believe that this technology will be pivotal in the development and advancement of microsensors and their biological applications.
In this paper, theories of particle removal by high-frequency ultrasonic waves are discussed and tested against recent experimental data. First, the principal adhesion forces such as van der Waals forces are briefly reviewed and the typical uncertainties in their size in particle-surface systems are assessed. The different ultrasound-induced forces-linear forces such as added mass, drag, lift, and Basset forces and nonlinear ones due to radiation pressure, and drag exerted by acoustic streaming-are discussed and their magnitudes are evaluated for typical cleaning operations. It is shown that high-frequency ultrasound can clean particles most effectively in media with properties like water because: (1) the wavelength can be made comparable to the particle radius to promote effective sound-particle interaction; (2) the viscous boundary layer is thin, minimizing particle "hide-out;" and (3) both the added mass and radiation pressure forces exceed typical adhesion forces at high frequencies. Based on these analyses, possible mechanisms of particle removal are discussed and interpreted in terms of experimental observations of particle cleaning.< >