Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design.

The analysis of low-abundance protein molecules in human serum is reported based on counting of the individual affinity-captured analyte on a solid sensor surface, yielding a readout format similar to digital assays. In this approach, a sandwich immunoassay with rolling circle amplification (RCA) is used for single molecule detection (SMD) through associating the target analyte with spatially distinct bright spots observed by fluorescence microscopy. The unspecific interaction of the target analyte and other immunoassay constituents with the sensor surface is of particular interest in this work, as it ultimately limits the performance of this assay. It is minimized by the design of the respective biointerface and thiol self-assembled monolayer with oligoethylene (OEG) head groups, and a poly[oligo(ethylene glycol) methacrylate] (pHOEGMA) antifouling polymer brush was used for the immobilization of the capture antibody (cAb) on the sensor surface. The assay relying on fluorescent postlabeling of long single-stranded DNA that are grafted from the detection antibody (dAb) by RCA was established with the help of combined surface plasmon resonance and surface plasmon-enhanced fluorescence monitoring of reaction kinetics. These techniques were employed for in situ measurements of conjugating of cAb to the sensor surface, tagging of short single-stranded DNA to dAb, affinity capture of the target analyte from the analyzed liquid sample, and the fluorescence readout of the RCA product. Through mitigation of adsorption of nontarget molecules on the sensor surface by tailoring of the antifouling biointerface, optimizing conjugation chemistry, and by implementing weak Coulombic repelling between dAb and the sensor surface, the limit of detection (LOD) of the assay was substantially improved. For the chosen interleukin-6 biomarker, SMD assay with LOD at a concentration of 4.3 fM was achieved for model (spiked) samples, and validation of the ability of detection of standard human serum samples is demonstrated.

Rapid prototyping of PMMA-based microfluidic spheroid-on-a-chip models using micromilling and vapour-assisted thermal bonding.

The application of microfluidic devices as next-generation cell and tissue culture systems has increased impressively in the last decades. With that, a plethora of materials as well as fabrication methods for these devices have emerged. Here, we describe the rapid prototyping of microfluidic devices, using micromilling and vapour-assisted thermal bonding of polymethyl methacrylate (PMMA), to create a spheroid-on-a-chip culture system. Surface roughness of the micromilled structures was assessed using scanning electron microscopy (SEM) and atomic force microscopy (AFM), showing that the fabrication procedure can impact the surface quality of micromilled substrates with milling tracks that can be readily observed in micromilled channels. A roughness of approximately 153 nm was created. Chloroform vapour-assisted bonding was used for simultaneous surface smoothing and bonding. A 30-s treatment with chloroform-vapour was able to reduce the surface roughness and smooth it to approximately 39 nm roughness. Subsequent bonding of multilayer PMMA-based microfluidic chips created a durable assembly, as shown by tensile testing. MDA-MB-231 breast cancer cells were cultured as multicellular tumour spheroids in the device and their characteristics evaluated using immunofluorescence staining. Spheroids could be successfully maintained for at least three weeks. They consisted of a characteristic hypoxic core, along with expression of the quiescence marker, p27kip1. This core was surrounded by a ring of Ki67-positive, proliferative cells. Overall, the method described represents a versatile approach to generate microfluidic devices compatible with biological applications.

Functionalized Terpolymer-Brush-Based Biointerface with Improved Antifouling Properties for Ultra-Sensitive Direct Detection of Virus in Crude Clinical Samples.

New analytical techniques that overcome major drawbacks of current routinely used viral infection diagnosis methods, i.e., the long analysis time and laboriousness of real-time reverse-transcription polymerase chain reaction (qRT-PCR) and the insufficient sensitivity of "antigen tests", are urgently needed in the context of SARS-CoV-2 and other highly contagious viruses. Here, we report on an antifouling terpolymer-brush biointerface that enables the rapid and sensitive detection of SARS-CoV-2 in untreated clinical samples. The developed biointerface carries a tailored composition of zwitterionic and non-ionic moieties and allows for the significant improvement of antifouling capabilities when postmodified with biorecognition elements and exposed to complex media. When deployed on a surface of piezoelectric sensor and postmodified with human-cell-expressed antibodies specific to the nucleocapsid (N) protein of SARS-CoV-2, it made possible the quantitative analysis of untreated samples by a direct detection assay format without the need of additional amplification steps. Natively occurring N-protein-vRNA complexes, usually disrupted during the sample pre-treatment steps, were detected in the untreated clinical samples. This biosensor design improved the bioassay sensitivity to a clinically relevant limit of detection of 1.3 × 104 PFU/mL within a detection time of only 20 min. The high specificity toward N-protein-vRNA complexes was validated both by mass spectrometry and qRT-PCR. The performance characteristics were confirmed by qRT-PCR through a comparative study using a set of clinical nasopharyngeal swab samples. We further demonstrate the extraordinary fouling resistance of this biointerface through exposure to other commonly used crude biological samples (including blood plasma, oropharyngeal, stool, and nasopharyngeal swabs), measured via both the surface plasmon resonance and piezoelectric measurements, which highlights the potential to serve as a generic platform for a wide range of biosensing applications.

Analyte transport to micro- and nano-plasmonic structures.

The study of optical affinity biosensors based on plasmonic nanostructures has received significant attention in recent years. The sensing surfaces of these biosensors have complex architectures, often composed of localized regions of high sensitivity (electromagnetic hot spots) dispersed along a dielectric substrate having little to no sensitivity. Under conditions such that the sensitive regions are selectively functionalized and the remaining regions passivated, the rate of analyte capture (and thus the sensing performance) will have a strong dependence on the nanoplasmonic architecture. Outside of a few recent studies, there has been little discussion on how changes to a nanoplasmonic architecture will affect the rate of analyte transport. We recently proposed an analytical model to predict transport to such complex architectures; however, those results were based on numerical simulation and to date, have only been partially verified. In this study we measure the characteristics of analyte transport across a wide range of plasmonic structures, varying both in the composition of their base plasmonic element (microwires, nanodisks, and nanorods) and the packing density of such elements. We functionalized each structure with nucleic acid-based bioreceptors, where for each structure we used analyte/receptor sequences as to maintain a Damköhler number close to unity. This method allows to extract both kinetic (in the form of association and dissociation constants) and analyte transport parameters (in the form of a mass transfer coefficient) from sensorgrams taken from each substrate. We show that, despite having large differences in optical characteristics, measured rates of analyte transport for all plasmonic structures match very well to predictions using our previously proposed model. These results highlight that, along with optical characteristics, analyte transport plays a large role in the overall sensing performance of a nanoplasmonic biosensor.

Microfluidic Analyte Transport to Nanorods for Photonic and Electrochemical Sensing Applications.

There has recently been a growing use of surface bound nanorods within electrochemical and optical sensing applications. Predictions of the microfluidic rate of analyte transport to such nanorods (either individual or to an array) remain important for sensor design and data analysis; however, such predictions are difficult, as nanorod aspect ratios can vary by several orders of magnitude. In this study, through the use of numerical simulation, we propose an explicit analytical approach to predict the steady-state diffusion-limited rate of mass transport to (individual) surface bound nanorods of variable aspect ratio. We show that, when compared to simulation, this approach provides accurate estimations across a wide range of Péclet numbers.

(Bio)Sensing Using Nanoparticle Arrays: On the Effect of Analyte Transport on Sensitivity.

There has recently been an extensive amount of work regarding the development of optical, electrical, and mechanical (bio)sensors employing planar arrays of surface-bound nanoparticles. The sensor output for these systems is dependent on the rate at which analyte is transported to, and interacts with, each nanoparticle in the array. There has so far been little discussion on the relationship between the design parameters of an array and the interplay of convection, diffusion, and reaction. Moreover, current methods providing such information require extensive computational simulation. Here we demonstrate that the rate of analyte transport to a nanoparticle array can be quantified analytically. We show that such rates are bound by both the rate to a single NP and that to a planar surface (having equivalent size as the array), with the specific rate determined by the fill fraction: the ratio between the total surface area used for biomolecular capture with respect to the entire sensing area. We characterize analyte transport to arrays with respect to changes in numerous parameters relevant to experiment, including variation of the nanoparticle shape and size, packing density, flow conditions, and analyte diffusivity. We also explore how analyte capture is dependent on the kinetic parameters related to an affinity-based biosensor, and furthermore, we classify the conditions under which the array might be diffusion- or reaction-limited. The results obtained herein are applicable toward the design and optimization of all (bio)sensors based on nanoparticle arrays.

Biosensor Enhancement Using Grooved Micromixers: Part II, Experimental Studies.

In this study we examine the experimental use of the staggered herringbone mixer (SHM) for the signal enhancement of a microfluidic surface plasmon resonance imaging (SPRi) affinity-based biosensor. We define the signal enhancement (Emix) as the ratio of the time-dependent slope of the sensor response of a SHM-based microfluidic channel and that of an unmixed channel; Emix is directly proportional to changes in the sensor sensitivity and inversely proportional to changes in the sensor limit of detection (LOD). Measurements were carried out for three SHM designs under a wide range of volumetric flow rates for two analytes: high diffusivity ssDNA and low diffusivity Escherichia coli bacteria. The experimental data collected in this study was found to exhibit a good match to that predicted by the numerical methods discussed in part I of this study. We found that Emix is dependent on the SHM groove geometry, the Péclet number Pe, and the overall microchannel length L; these dependencies are discussed in detail. For realistic experimental conditions, the enhancement that the SHM can provide is in the range of 1 < Emix < 5 (0% < improvement < 400%).

Biosensor enhancement using grooved micromixers: part I, numerical studies.

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 a biosensor surface. We 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.

Biosensing enhancement using passive mixing structures for microarray-based sensors.

The combination of microarray technologies with microfluidic sample delivery and real-time detection methods has the capability to simultaneously monitor 10-1000 s of biomolecular interactions in a single experiment. Despite the benefits that microfluidic systems provide, they typically operate in the laminar flow regime under mass transfer limitations, where large analyte depletion layers act as a resistance to analyte capture. By locally stirring the fluid and delivering fresh analyte to the capture spot, the use of passive mixing structures in a microarray environment can reduce the negative effects of these depletion layers and enhance the sensor performance. Despite their large potential, little attention has been given to the integration of these mixing structures in microarray sensing environments. In this study, we use passive mixing structures to enhance the mass transfer of analyte to a capture spot within a microfluidic flow cell. Using numerical methods, different structure shapes and heights were evaluated as means to increase local fluid velocities, and in turn, rates of mass transfer to a capture spot. These results were verified experimentally via the real-time detection of 20-mer ssDNA for an array of microspots. Both numerical and experimental results showed that a passive mixing structure situated directly over the capture spot can significantly enhance the binding rate of analyte to the sensing surface. Moreover, we show that these structures can be used to enhance mass transfer in experiments regarding an array of capture spots. The results of this study can be applied to any experimental system using microfluidic sample delivery methods for microarray detection techniques.

Enhancement of affinity-based biosensors: effect of sensing chamber geometry on sensitivity.

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 J ≈ H(-2/3). 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.

Spatially resolved electrochemical sensing of chemical gradients.

Chemical gradients drive a diverse set of biological processes ranging from nerve transduction to ovulation. At present, the most common method for quantifying chemical gradients is microscopy. Here, a new concept for probing spatial and temporal chemical gradients is reported that uses a multi-layer microfluidic device to measure analyte concentration as a function of lateral position in a microfluidic channel using electrochemistry in a format that is readily adaptable to multi-analyte sensing.

Mapping spatiotemporal molecular distributions using a microfluidic array.

The spatial and temporal distributions of an extensive number of diffusible molecules drive a variety of complex functions. These molecular distributions often possess length scales on the order of a millimeter or less; therefore, microfluidic devices have become a powerful tool to study the effects of these molecular distributions in both chemical and biological systems. Although there exist a number of studies utilizing microdevices for the creation of molecular gradients, there are few, if any, studies focusing on the measurement of spatial and temporal distributions of molecular species created within the study system itself. Here we present a microfluidic device capable of sampling multiple chemical messengers in a spatiotemporally resolved manner. This device operates through spatial segregation of nanoliter-sized volumes of liquid from a primary sample reservoir into a series of analysis microchannels, where fluid pumping is accomplished via a system of passive microfluidic pumps. Subsequent chemical analysis within each microchannel, achieved via optical or bioanalytical methods, yields quantitative data on the spatial and temporal information for any analytes of interest existing within the sample reservoir. These techniques provide a simple, cost-effective route to measure the spatiotemporal distributions of molecular analytes, where the system can be tailored to study both chemical and biological systems.

An optical waveguide array biosensor for serology - biomed 2010.

A label-free optical waveguide immunosensor was designed, fabricated and tested. Different from other popular resonance-based biosensors, such as surface-plasmon-resonance (SPR) or ring/disk resonance biosensors, the local evanescent array coupled (LEAC) biosensor relies on a local evanescent field shift mechanism and can be readily manufactured using trailing-edge integrated-circuit technology with chip-scale microfluidics technology to provide very low cost. The anticipated final form of the sensor technology will require no external equipment enabling disposable use for point-of-care disease detection in non-traditional health-care settings. The local detection ability enables the LEAC biosensor the capability to detect and identify multiple analytes on the same waveguide. On-chip detection is accomplished by integrating buried polysilicon detector arrays under a silicon nitride waveguide. Observations of antibody-antigen interactions using the LEAC biosensor are reported.

Passive microfluidic pumping using coupled capillary/evaporation effects.

Controlled pumping of fluids through microfluidic networks is a critical unit operation ubiquitous to lab-on-a-chip applications. Although there have been a number of studies involving the creation of passive flows within lab-on-a-chip devices, none has shown the ability to create temporally stable flows for periods longer than several minutes. Here a passive pumping approach is presented in which a large pressure differential arising from a small, curved meniscus situated along the bottom corners of an outlet reservoir serves to drive fluid through a microfluidic network. The system quickly reaches steady-state and is able to provide precise volumetric flow rates for periods lasting over an hour. A two-step mathematical model provides accurate predictions of fluid and mass transport dynamics in these devices, as validated by particle tracking in laboratory systems. Precise flow rates spanning an order of magnitude are accomplished via control of the microchannel and outlet reservoir dimensions. This flow mechanism has the potential to be applied to many micro-total analytical system devices that utilize pressure-driven flow; as an illustrative example, the pumping technique is applied for the passive generation of temporally stable chemical gradients.

Evaporation from microreservoirs.

As a result of very large surface area to volume ratios, evaporation is of significant importance when dealing with lab-on-a-chip devices that possess open air/liquid interfaces. For devices utilizing a reservoir as a fluid delivery method to a microfluidic network, excessive evaporation can quickly lead to reservoir dry out and overall device failure. Predicting the rates of evaporation from these reservoirs is difficult because the position of the air/liquid interface changes with time as the volume of liquid in the reservoir decreases. Here we present a two-step method to accurately predict the rates of evaporation of such an interface over time. First, a simple method is proposed to determine the shape of an air/liquid meniscus in a reservoir given a specific liquid volume. Second, computational fluid dynamics simulations are used to calculate the instantaneous rate of evaporation for that meniscus shape. It is shown that the rate of evaporation is strongly dependent on the overall geometry of the system, enhanced in expanding reservoirs while suppressed in contracting reservoirs, where the geometry can be easily controlled with simple experimental methods. Using no adjustable parameters, the model accurately predicts the position of the inner moving contact line as a function of time following meniscus rupture in poly(dimethylsiloxane) reservoirs, and predicts the overall time for the persistence of liquid in those reservoirs to within 0.5 minutes. The methods in this study can be used to design holding reservoirs for lab-on-a-chip devices that involve no external control of evaporation, such that evaporation rates can be adjusted as necessary by modification of the reservoir geometry.

Geometrical optimization of helical flow in grooved micromixers.

Owing to the enhancement of surface effects at the micro-scale, patterned grooves on a micro-channel floor remain a powerful method to induce helical flows within a pressure driven system. Although there have been a number of numerical studies on geometrical effects concerning fluid mixing within the staggered herringbone mixer, all have focused mainly on the groove angle and depth, two factors that contribute greatly to the magnitude of helical flow. Here we present a new geometrical factor that significantly affects the generation of helical flow over patterned grooves. By varying the ratio of the length of the grooves to the neighboring ridges, helical flow can be optimized for a given groove depth and channel aspect ratio, with up to 50% increases in transverse flow possible. A thorough numerical study of over 700 cases details the magnitude of helical flow over unsymmetrical patterned grooves in a slanted groove micro-mixer, where the optimized parameters for the slanted groove mixer can be translated to the staggered herringbone mixer. The optimized groove geometries are shown to have a large dependence on the channel aspect ratio, the groove depth ratio, and the ridge length.