Double-Sided Tape in Microfluidics: A Cost-Effective Method in Device Fabrication.

The demand for easy-to-use, affordable, accessible, and reliable technology is increasing in biological, chemical, and medical research. Microfluidic devices have the potential to meet these standards by offering cost-effective, highly sensitive, and highly specific diagnostic tests with rapid performance and minimal sample volumes. Traditional microfluidic device fabrication methods, such as photolithography and soft lithography, are time-consuming and require specialized equipment and expertise, making them costly and less accessible to researchers and clinicians and limiting the applicability and potential of microfluidic devices. To address this, researchers have turned to using new low-cost materials, such as double-sided tape for microfluidic device fabrication, which offers simple and low-cost processes. The innovation of low-cost and easy-to-make microfluidic devices improves the potential for more devices to be transitioned from laboratories to commercialized products found in stores, offices, and homes. This review serves as a comprehensive summary of the growing interest in and use of double-sided tape-based microfluidic devices in the last 20 years. It discusses the advantages of using double-sided tape, the fabrication techniques used to create and bond microfluidic devices, and the limitations of this approach in certain applications.

Rapid detection of vaccinia virus using biofunctionalized fiber-optic ball-tip biosensors.

In this work, we present the development and biofunctionalization of a fiber-optic ball-resonator biosensor for the real-time detection of vaccinia poxvirus. We fabricated several ball-tip resonators, functionalized through a silanization process to immobilize two bioreceptors: the monoclonal anti-L1R antibody targeting the L1R protein, and the polyclonal rabbit serum antibodies targeting the whole vaccinia virus (VV) pathogen. Experimental measurements were carried out to detect VV in concentrations from 103 to 108 plaque-forming units (PFU), with a limit of detection of around 1.7-4.3 × 103 PFU and a log-quadratic pattern, with a response up to 5 × 10-4 RIU (refractive index units). The specificity was assessed against herpes simplex virus, used as a non-specific control, with the best results obtained with anti-L1R monoclonal antibodies, and through the detection of vaccinia virus/herpes simplex-1 combination. The obtained results provide a real-time viral recognition with a label-free sensing platform, having rapid response and ease of manufacturing, and paving the road to the seamless detection of poxviruses affecting different human and animal species using optical fibers.

Optimizing an Optical Cavity-Based Biosensor for Enhanced Sensitivity.

The rapid advancement of biosensor technology has revolutionized healthcare, offering improved sensitivity, specificity, and portability. We have developed an optical cavity-based biosensor (OCB) as a promising solution due to its label-free detection, high sensitivity, real-time monitoring, multiplexing capability, and versatility. The OCB consists of an optical cavity structure (OCS), optical components, and a low-cost camera. The OCS is created by two partially reflective surfaces separated by a small gap, where the interaction between target analytes and immobilized receptors leads to a shift in the resonance transmission spectrum, caused by minute changes in the local refractive index (RI). In our previous work, we successfully detected these small changes with a simple OCS and cost-effective components using a differential detection method. Building upon these achievements, this study focuses on optimizing the OCS, improving the camera settings, and enhancing the differential detection approach. By increasing the reflectance of the surfaces and optimizing the optical cavity widths correspondingly, we achieved an improved limit of detection (LOD). We also investigated how the charge-coupled device (CCD) camera shutter time affects the LOD. Additionally, we introduced a new differential equation to further enhance the sensitivity of our system. Through these advancements, we could improve the LOD of the OCB by 7.2 times, specifically for an OCS with a cavity thickness of 9.881 µm and a silver thickness of 46.87 nm. These findings not only contribute to the ongoing effort of optimizing the OCB, but also pave the way for the development of advanced point-of-care biosensors with enhanced detection capabilities.

Spatial-Division Multiplexing Approach for Simultaneous Detection of Fiber-Optic Ball Resonator Sensors: Applications for Refractometers and Biosensors.

Fiber-optic ball resonators are an attractive technology for refractive index (RI) sensing and optical biosensing, as they have good sensitivity and allow for a rapid and repeatable manufacturing process. An important feature for modern biosensing devices is the multiplexing capacity, which allows for interrogating multiple sensors (potentially, with different functionalization methods) simultaneously, by a single analyzer. In this work, we report a multiplexing method for ball resonators, which is based on a spatial-division multiplexing approach. The method is validated on four ball resonator devices, experimentally evaluating both the cross-talk and the spectral shape influence of one sensor on another. We show that the multiplexing approach is highly efficient and that a sensing network with an arbitrary number of ball resonators can be designed with reasonable penalties for the sensing capabilities. Furthermore, we validate this concept in a four-sensor multiplexing configuration, for the simultaneous detection of two different cancer biomarkers across a widespread range of concentrations.

A Novel ssDNA Aptamer Targeting Carcinoembryonic Antigen: Selection and Characterization.

One of the major causes of a drastically shorter life expectancy and one of the most prevalent diseases in the world today is cancer. Given the data on the rise in cancer cases throughout the world, it is obvious that, despite the diagnostic techniques currently being used, there is a pressing need to develop precise and sensitive techniques for early diagnosis of the disease. A high degree of affinity and specificity towards particular targets is maintained by the short nucleic acid molecules known as aptamers. Aptamers outperform antibodies due to their unique benefits, such as their simplicity in synthesis and modification, lack of toxicity, and long-term stability. Utilizing an accurate recognition element and a robust signal transduction mechanism, molecular diagnostics can be extremely sensitive and specific. In this study, development of new single-stranded DNA aptamers against CEA for use in cancer diagnostics was accomplished using SELEX and NGS methods. As a result of 12 iterative SELEX rounds, nine aptamer candidates against CEA were developed. NGS comparative analysis revealed that round twelve had an enriched number of aptamers that were specifically bound, as opposed to round eight. Among the selected nine sequences characterized by bioinformatics analysis and ELONA, an aptamer sequence with the highest specificity and affinity for the target protein was identified and further examined. Aptamer sequence (6) was screened in a concentration-dependent assay, specificity analysis was performed, and its potential secondary and tertiary structures were predicted, which enabled us to test one of the possible putative interactions with CEA. Finally, aptamer sequence (6) labelled with a Cy5 fluorescent tag was used in confocal microscopy to observe its binding towards the CEA expressed in HT-29 human colon adenocarcinoma cell line.

Optimization of Cladding Diameter for Refractive Index Sensing in Tilted Fiber Bragg Gratings.

This work presents an experimental investigation of the effect of chemical etching on the refractive index (RI) sensitivity of tilted fiber Bragg gratings (TFBGs). Hydrofluoric acid (HF) was used stepwise in order to reduce the optical fiber diameter from 125 µm to 13 µm. After each etching step, TFBGs were calibrated using two ranges of RI solutions: the first one with high RI variation (from 1.33679 RIU to 1.37078 RIU) and the second with low RI variation (from 1.34722 RIU to 1.34873 RIU). RI sensitivity was analyzed in terms of wavelength shift and intensity change of the grating resonances. The highest amplitude sensitivities obtained are 1008 dB/RIU for the high RI range and 8160 dB/RIU for the low RI range, corresponding to the unetched TFBG. The highest wavelength sensitivities are 38.8 nm/RIU for a fiber diameter of 100 µm for the high RI range, and 156 nm/RIU for a diameter of 40 µm for the small RI range. In addition, the effect of the etching process on the spectral intensity of the cladding modes, their wavelength separation and sensor linearity (R2) were studied as well. As a result, an optimization of the etching process is provided, so that the best trade-off between sensitivity, intensity level, and fiber thickness can be obtained.

Ultralow Limit Detection of Soluble HER2 Biomarker in Serum with a Fiber-Optic Ball-Tip Resonator Assisted by a Tilted FBG.

An optical-fiber biosensor has been developed for the detection of the breast cancer biomarker soluble human epidermal growth factor receptor-2 (sHER2). The sensor was fabricated by combining a tilted fiber Bragg grating (TFBG) with a ball resonator, allowing us to achieve an excellent sensitivity compared to other optical-fiber-based sensors. The sensor exhibits a resonance comb excited by the TFBG and the spectral profile of the ball resonator. The detection of sHER2 at extremely low concentrations was carried out by tracking the amplitude change of selected resonances. The therapeutic anti-HER2 monoclonal antibody Trastuzumab has been used to functionalize the biosensor with silane surface chemistry. The sensor features a sensitivity of 4034 dB/RIU with a limit of detection (LoD) in buffer and in a 1/10 diluted serum of 151.5 ag/mL and 3.7 pg/mL, respectively. At relatively high protein concentrations (64 ng/mL) binding to sHER (7.36 dB) as compared to control proteins (below 0.7 dB) attested the high specificity of sHER2 detection.

Review: 3-Aminopropyltriethoxysilane (APTES) Deposition Methods on Oxide Surfaces in Solution and Vapor Phases for Biosensing Applications.

Surface functionalization and bioreceptor immobilization are critical processes in developing a highly sensitive and selective biosensor. The silanization process with 3-aminopropyltriethoxysilane (APTES) on oxide surfaces is frequently used for surface functionalization because of beneficial characteristics such as its bifunctional nature and low cost. Optimizing the deposition process of the APTES layer to obtain a monolayer is crucial to having a stable surface and effectively immobilizing the bioreceptors, which leads to the improved repeatability and sensitivity of the biosensor. This review provides an overview of APTES deposition methods, categorized into the solution-phase and vapor-phase, and a comprehensive summary and guide for creating stable APTES monolayers on oxide surfaces for biosensing applications. A brief explanation of APTES is introduced, and the APTES deposition methods with their pre/post-treatments and characterization results are discussed. Lastly, APTES deposition methods on nanoparticles used for biosensors are briefly described.

Perspectives on Assembling Coronavirus Spikes on Fiber Optics to Reveal Broadly Recognizing Antibodies and Generate a Universal Coronavirus Detector.

In time of COVID-19 biological detection technologies are of crucial relevance. We propose here the use of state of the art optical fiber biosensors to address two aspects of the fight against SARS-CoV-2 and other pandemic human coronaviruses (HCoVs). Fiber optic biosensors functionalized with HCoV spikes could be used to discover broadly neutralizing antibodies (bnAbs) effective against known HCoVs (SARS-CoV, MERS-CoV and SARS-CoV-2) and likely future ones. In turn, identified bnAbs, once immobilized onto fiber optic biosensors, should be capable to detect HCoVs as diagnostic and environmental sensing devices. The therapeutic and preventative value of bnAbs is immense as they can be used for passive immunization and for the educated development of a universal vaccine (active immunization). Hence, HCoV bnAbs represent an extremely important resource for future preparedness against coronavirus-borne pandemics. Furthermore, the assembly of bnAb-based biosensors constitutes an innovative approach to counteract public health threats, as it bears diagnostic competence additional to environmental detection of a range of pandemic strains. This concept can be extended to different pandemic viruses, as well as bio-warfare threats that entail existing, emerging and extinct viruses (e.g., the smallpox-causing Variola virus). We report here the forefront fiber optic biosensor technology that could be implemented to achieve these aims.

Fiber Optic Refractive Index Sensors Based on a Ball Resonator and Optical Backscatter Interrogation.

In this work, we introduced fabrication and interrogation of simple and highly sensitive fiber-optic refractive index (RI) sensors based on ball resonators built on the tip of single-mode fibers. The probes have been fabricated through a CO2 fiber splicer, with a fast (~600 s) and repeatable method. The ball resonator acted as a weak interferometer with a return loss below -50 dB and was interrogated with an optical backscatter reflectometer measuring the reflection spectrum. The ball resonators behaved as weak interferometers with a shallow fringe and a spectrum that appeared close to a random signal, and RI sensitivity could be measured either through wavelength shift or amplitude change. In this work, we reported four samples having sensitivity ranges 48.9-403.3 nm/RIU and 256.0-566.2 dB/RIU (RIU = refractive index unit). Ball resonators appeared as a sensitive and robust platform for RI sensing in liquid and can be further functionalized for biosensing.

Reflector-less nanoparticles doped optical fiber biosensor for the detection of proteins: Case thrombin.

A miniature biosensing platform based on MgO-based nanoparticle doped optical fiber was developed for the biomolecule detection. The technology used a single mode fiber with MgO-based nanoparticles doped core. The detection was based on collecting the Rayleigh backscattering signatures with increased gain upon the etching of the fiber 1-2 mm away from the tip. The shift from the backscattered signal with the maximum value of the cross-correlation was used to report the results. The sensor exhibited a sensitivity range from 0.75 nm/refractive index unit up to 19.63 nm/refractive index unit for a refractive index range from 1.3329 up to 1.37649. The deposition of the thin gold layer increased the overall sensitivity of the biosensor by 3.7 times for the etched part of the fiber with diameter 8-9 µm. The proposed biosensor was tested for the detection of thrombin molecule concentrations ranging from 0.625 µg/ml to 20 µg/ml. Thiol modified DNA specific aptamers were used to functionalize the gold coated surface of the fiber for the detection. The sensor showed detectable sensitivity and specificity as compared to the other control proteins. The proposed biosensing platform could be multiplexed and can be used in vivo for the detection in clinical settings due to its miniature size, biocompatibility of silica glass and reflector less set up.

An aptasensor for the detection of Mycobacterium tuberculosis secreted immunogenic protein MPT64 in clinical samples towards tuberculosis detection.

This work presents experimental results on detection of Mycobacterium tuberculosis secreted protein MPT64 using an interdigitated electrode (IDE) which acts as a platform for capturing an immunogenic protein and an electrochemical impedance spectroscopy (EIS) as a detection technique. The assay involves a special receptor, single stranded DNA (ssDNA) aptamer, which specifically recognizes MPT64 protein. The ssDNA immobilization on IDE was based on a co-adsorbent immobilization at an optimized ratio of a 1/100 HS-(CH6)6-OP(O)2O-(CH2CH2O)6-5'-TTTTT-aptamer-3'/6-mercaptohexanol. The optimal sample incubation time required for a signal generation on an aptamer modified IDE was found to be at a range of 15-20 min. Atomic Force Microscopy (AFM) results confirmed a possible formation of an aptamer - MPT64 complex with a 20 nm roughness on the IDE surface vs. 4.5 nm roughness for the IDE modified with the aptamer only. A limit of detection for the EIS aptasensor based on an IDE for the detection of MPT64 in measurement buffer was 4.1 fM. The developed EIS aptasensor was evaluated on both serum and sputum clinical samples from the same TB (-) and TB (+) patients having a specificity and sensitivity for the sputum sample analysis 100% and 76.47%, respectively, and for the serum sample analysis 100% and 88.24%, respectively. The developed aptasensor presents a sensitive method for the TB diagnosis with the fast detection time.

Functionalized etched tilted fiber Bragg grating aptasensor for label-free protein detection.

An aptasensor based on etched tilted fiber Bragg grating (eTFBG) is developed on a single-mode optical fiber targeting biomolecule detection. TFBGs were chemically etched using hydrofluoric acid (HF) to partially remove the fiber cladding. The sensor response was coarsely interrogated, resulting on a sensitivity increase from 1.25 nm/RIU (refractive index unit) at the beginning of the process, up to 23.38 nm/RIU at the end of the etching, for a RI range from 1.3418 to 1.4419 RIU. The proposed aptasensor showed improved RI sensitivity as compared to the unetched TFBG, without requiring metal depositions on the fiber surface or polarization control during the measurements. The proposed sensor was tested for the detection of thrombin-aptamer interactions based on silane-coupling surface chemistry, with thrombin concentrations ranging from 2.5 to 40 nM. Functionalized eTFBGs provided a competitive platform for biochemical interaction measurements, showing sensitivity values ranging from 2.3 to 3.3 p.m./nM for the particular case of thrombin detection.

Electrochemical aptasensor using optimized surface chemistry for the detection of Mycobacterium tuberculosis secreted protein MPT64 in human serum.

Tuberculosis (TB) remains one of the leading causes of mortality worldwide. There is a great need for the development of diagnostic tests, which are reliable, sensitive, stable, and low cost to enable early diagnosis of TB in communities with scarce resources. This study reports the optimization and evaluation of a synthetic receptor, an aptamer, for the detection of the secreted protein MPT64, which is a highly immunogenic polypeptide of Mycobacterium tuberculosis, a causative agent of TB. The study investigates combinatorial effects of an aptamer linker and a co-adsorbent onto a gold electrode for optimal binding efficiency and reduced non-specific interactions for label-free detection of MPT64 using electrochemical impedance spectroscopy. Two types of co-adsorbents and two types of aptamer linkers were studied and high specificity and sensitivity to MPT64 was observed for a surface prepared with a thiol PEGylated aptamer HS-(CH2)6-OP(O)2O-(CH2CH2O)6-TTTTT-aptamer and 6-mercaptohexanol in a ratio of 1:100. The developed aptamer-based sensor was successfully used with spiked human serum sample with a limit of detection of 81 pM This work demonstrates the use of the MPT64 aptamer as a lower cost, more sustainable and stable alternative of antibodies for the development of point-of-care TB biosensors decreasing the detection time from several days or hours to thirty minutes.

Wavelet-Based Demodulation of Multimode Etched Fiber Bragg Grating Refractive Index Sensor.

Etched fiber Bragg grating (EFBG)-based sensors are used as evanescent field sensors for refractive index detection. When the fiber thickness is thin and the refractive index sensitivity increases, the number of propagating modes increases, resulting in a spectral enlargement that complicates the interrogation of the sensor. In this work, we present a method to analyze the spectrum of a multimode etched fiber Bragg grating (MMEFBG) in the wavelet domain, which analyzes the amount of spectral density independently from the peak reflectivity value. The proposed interrogation method permits defining the integral of the spectral density as a novel and unconventional estimator. With respect to the conventional estimators based on wavelength shift, this estimator can better exploit the larger amount of information given by the spectral enlargement typical of multimode behavior. Results were obtained by etching an MMEFBG in hydrofluoric acid and using water/sucrose mixtures to evaluate the refractive index sensitivity, validating the interrogation method.

Fiber optic refractive index sensors through spectral detection of Rayleigh backscattering in a chemically etched MgO-based nanoparticle-doped fiber.

We demonstrate and experimentally validate a fiber optic refractive index (RI) sensor obtained by simply etching a high-scattering MgO-based nanoparticle-doped single-mode fiber in hydrofluoric acid (HF). The fiber has 32.3 dB stronger Rayleigh scattering than a standard fiber, allowing a detection of scattering spectral signatures with an optical backscatter reflectometer, even when the core is exposed to the outer RI. The obtained sensitivity is 1.53 nm/RIU (RI units), measured by correlating the scattering spectra. We prove the possibility of implementing a distributed RI detection (seven locations spaced by 1 mm). The fabrication method for this RI sensor is simplified, since it simply requires etching in an HF bath, without the need of inscribing reflective elements or fabricating microstructures in the fiber.

Etched Fiber Bragg Grating Biosensor Functionalized with Aptamers for Detection of Thrombin.

A biosensor based on an etched Fiber Bragg Grating (EFBG) for thrombin detection is reported. The sensing system is based on a Fiber Bragg Grating (FBG) with a Bragg wavelength of 1550 nm, wet-etched in hydrofluoric acid (HF) for ~27 min, to achieve sensitivity to a refractive index (RI) of 17.4 nm/RIU (refractive index unit). Subsequently, in order to perform a selective detection of thrombin, the EFBG has been functionalized with silane-coupling agent 3-(aminopropyl)triethoxysilane (APTES) and a cross-linker, glutaraldehyde, for the immobilization of thrombin-binding aptamer. The biosensor has been validated for thrombin detection in concentrations ranging from 10 nM to 80 nM. The proposed sensor presents advantages with respect to other sensor configurations, based on plasmonic resonant tilted FBG or Long Period Grating (LPG), for thrombin detection. Firstly, fabricating an EFBG only requires chemical etching. Moreover, the functionalization method used in this study (silanization) allows the avoidance of complicated and expensive fabrications, such as thin film sputtering or chemical vapor deposition. Due to their characteristics, EFBG sensors are easier to multiplex and can be used in vivo. This opens new possibilities for the detection of thrombin in clinical settings.

Partially etched chirped fiber Bragg grating (pECFBG) for joint temperature, thermal profile, and refractive index detection.

In this work, a partially etched chirped fiber Bragg grating (pECFBG) is introduced, as a compact sensor for multi-parametric measurement of temperature, thermal gradients over the active length, and refractive index. The sensor is fabricated by wet-etching a portion of a 14-mm linearly chirped FBG with linear chirp profile. The resulting device has two active areas: the unetched part of the grating (2 mm) can be used either as a uniform temperature sensor, or to detect thermal gradients experienced through the grating length by means of a spectral reconstruction technique; the etched part (12 mm), besides having a similar thermal sensitivity, is exposed to refractive index sensing through the introduction of a sensitivity to external refractive index. Overall, the pECFBG structure behaves as a compact sensor with multi-parameter capability, that can both measure temperature and refractive index on the same grating, but also spatially resolve temperature detection through the grating section. The results have been validated through both a model and experimental setup, showing that the mutual correlation algorithm applied to different spectral parts of the grating is able to discriminate between uniform and gradient-shaped temperature profiles, and refractive index changes.

CFD Modeling of Chamber Filling in a Micro-Biosensor for Protein Detection.

Tuberculosis (TB) remains one of the main causes of human death around the globe. The mortality rate for patients infected with active TB goes beyond 50% when not diagnosed. Rapid and accurate diagnostics coupled with further prompt treatment of the disease is the cornerstone for controlling TB outbreaks. To reduce this burden, the existing gap between detection and treatment must be addressed, and dedicated diagnostic tools such as biosensors should be developed. A biosensor is a sensing micro-device that consists of a biological sensing element and a transducer part to produce signals in proportion to quantitative information about the binding event. The micro-biosensor cell considered in this investigation is designed to operate based on aptamers as recognition elements against Mycobacterium tuberculosis secreted protein MPT64, combined in a microfluidic-chamber with inlet and outlet connections. The microfluidic cell is a miniaturized platform with valuable advantages such as low cost of analysis with low reagent consumption, reduced sample volume, and shortened processing time with enhanced analytical capability. The main purpose of this study is to assess the flooding characteristics of the encapsulated microfluidic cell of an existing micro-biosensor using Computational Fluid Dynamics (CFD) techniques. The main challenge in the design of the microfluidic cell lies in the extraction of entrained air bubbles, which may remain after the filling process is completed, dramatically affecting the performance of the sensing element. In this work, a CFD model was developed on the platform ANSYS-CFX using the finite volume method to discretize the domain and solving the Navier-Stokes equations for both air and water in a Eulerian framework. Second-order space discretization scheme and second-order Euler Backward time discretization were used in the numerical treatment of the equations. For a given inlet-outlet diameter and dimensions of an in-house built cell chamber, different inlet liquid flow rates were explored to determine an appropriate flow condition to guarantee an effective venting of the air while filling the chamber. The numerical model depicted free surface waves as promoters of air entrainment that ultimately may explain the significant amount of air content in the chamber observed in preliminary tests after the filling process is completed. Results demonstrated that for the present design, against the intuition, the chamber must be filled with liquid at a modest flow rate to minimize free surface waviness during the flooding stage of the chamber.

Selection, characterization, and application of DNA aptamers for detection of Mycobacterium tuberculosis secreted protein MPT64.

Rapid detection of Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis (TB), is important for global control of this disease. Aptamers have emerged as a potential rival for antibodies in therapeutics, diagnostics and biosensing due to their inherent characteristics. The aim of the current study was to select and characterize single-stranded DNA aptamers against MPT64 protein, one of the predominant secreted proteins of Mtb pathogen. Aptamers specific to MPT64 protein were selected in vitro using systematic evolution of ligands through exponential enrichment (SELEX) method. The selection was started with a pool of ssDNA library with randomized 40-nucleotide region. A total of 10 cycles were performed and seventeen aptamers with unique sequences were identified by sequencing. Dot Blot analysis was performed to monitor the SELEX process and to conduct the preliminary tests on the affinity and specificity of aptamers. Enzyme linked oligonucleotide assay (ELONA) showed that most of the aptamers were specific to the MPT64 protein with a linear correlation of R2 = 0.94 for the most selective. Using Surface Plasmon Resonance (SPR), dissociation equilibrium constant KD of 8.92 nM was obtained. Bioinformatics analysis of the most specific aptamers revealed the existence of a conserved as well as distinct sequences and possible binding site on MPT64. The specificity was determined by testing non-target ESAT-6 and CFP-10. Negligible cross-reactivity confirmed the high specificity of the selected aptamer. The selected aptamer was further tested on clinical sputum samples using ELONA and had sensitivity and specificity of 91.3% and 90%, respectively. Microscopy, culture positivity and nucleotide amplification methods were used as reference standards. The aptamers studied could be further used for the development of medical diagnostic tools and detection assays for Mtb.