Search results for: biosensing

Authors: Susmita Roy, Madhavan Nallani

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In recent years' polymersomes, self-assembled polymeric vesicles emerge from block copolymers, have been widely investigated due to their enhance stability and unique advantageous properties compared to their phospholipid counterpart, liposomes, dendrimers, and micelles. It provides a distinctive platform for advanced therapeutics and the creation of complex (bio) catalytically active systems for research in Nanomedicine and synthetic biology. Inspired by nature, where compartmentalization of biological components is all ubiquitous, we are interested in developing a platform technology of self-assembled multifunctional compartments with applications in areas from targeted drug/gene delivery, biosensing, pharmaceutical to cosmetics. Polymersome surfaces can be a proper choice of derivatization with a controlled amount of functional groups. To achieve site-specific targeting of polymersomes, biological recognition motives can be attached to the polymersomes surface by standard bioconjugation techniques, (like esterification, amidation, thiol-maleimide coupling, click-chemistry routes or other coupling methods). Herein, we are developing easy going, one-step bioconjugation strategies for site-specific surface functionalized biodegradable polymeric and/or polymer-lipid hybrid vesicles for targeted drug delivery. Biodegradable polymer, polycaprolactone-b-polyethylene glycol (PCL-PEG), polylactic acid-b-polyethylene glycol (PLA-PEG) and phospholipid, 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC) has been widely used for numerous vesicle formulations. Some of these drug-loaded formulations are being tested on mice for controlled release. These surface functionalized polymersomes are also appropriate for membrane protein reconstitution/insertion, antibodies conjugation and various bioconjugation with diverse targeted molecules for controlled drug delivery.

Keywords: drug delivery, membrane protein, polymersome, surface modification

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Authors: Hwang Ahreum, Kang Taejoon, Kim Bongsoo

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Nanosensors for high sensitive detection of diseases have been widely studied to improve the quality of life. Here, we suggest robust nano-plasmonic diagnostic sensor using cysteine tagged protein G (Cys3-protein G) and ultraflat, ultraclean and single-crystalline Au nanoplates. Protein G formed on an ultraflat Au surface provides ideal background for dense and uniform immobilization of antibodies. The Au is highly stable in diverse biochemical environment and can immobilize antibodies easily through Au-S bonding, having been widely used for various biosensing applications. Especially, atomically smooth single-crystalline Au nanomaterials synthesized using chemical vapor transport (CVT) method are very suitable to fabricate reproducible sensitive sensors. As the C-reactive protein (CRP) is a nonspecific biomarker of inflammation and infection, it can be used as a predictive or prognostic marker for various cardiovascular diseases. Cys3-protein G immobilized uniformly on the Au nanoplate enable CRP antibody (anti-CRP) to be ordered in a correct orientation, making their binding capacity be maximized for CRP detection. Immobilization condition for the Cys3-protein G and anti-CRP on the Au nanoplate is optimized visually by AFM analysis. Au nanoparticle - Au nanoplate (NPs-on-Au nanoplate) assembly fabricated from sandwich immunoassay for CRP can reduce zero-signal extremely caused by nonspecific bindings, providing a distinct surface-enhanced Raman scattering (SERS) enhancement still in 10-18 M of CRP concentration. Moreover, the NP-on-Au nanoplate sensor shows an excellent selectivity against non-target proteins with high concentration. In addition, comparing with control experiments employing a Au film fabricated by e-beam assisted deposition and linker molecule, we validate clearly contribution of the Au nanoplate for the attomolar sensitive detection of CRP. We expect that the devised platform employing the complex of single-crystalline Au nanoplates and Cys3-protein G can be applied for detection of many other cancer biomarkers.

Keywords: Au nanoplate, biomarker, diagnostic sensor, protein G, SERS

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Authors: Lorico D. S. Lapitan Jr., Yihan Xu, Yuan Guo, Dejian Zhou

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The ability of ultrasensitive detection of specific genes and discrimination of single nucleotide polymorphisms is important for clinical diagnosis and biomedical research. Herein, we report the development of a new ultrasensitive approach for label-free DNA detection using magnetic nanoparticle (MNP) assisted rapid target capture/separation in combination with signal amplification using poly-enzyme tagged polymer nanobead. The sensor uses an MNP linked capture DNA and a biotin modified signal DNA to sandwich bind the target followed by ligation to provide high single-nucleotide polymorphism discrimination. Only the presence of a perfect match target DNA yields a covalent linkage between the capture and signal DNAs for subsequent conjugation of a neutravidin-modified horseradish peroxidase (HRP) enzyme through the strong biotin-nuetravidin interaction. This converts each captured DNA target into an HRP which can convert millions of copies of a non-fluorescent substrate (amplex red) to a highly fluorescent product (resorufin), for great signal amplification. The use of polymer nanobead each tagged with thousands of copies of HRPs as the signal amplifier greatly improves the signal amplification power, leading to greatly improved sensitivity. We show our biosensing approach can specifically detect an unlabeled DNA target down to 10 aM with a wide dynamic range of 5 orders of magnitude (from 0.001 fM to 100.0 fM). Furthermore, our approach has a high discrimination between a perfectly matched gene and its cancer-related single-base mismatch targets (SNPs): It can positively detect the perfect match DNA target even in the presence of 100 fold excess of co-existing SNPs. This sensing approach also works robustly in clinical relevant media (e.g. 10% human serum) and gives almost the same SNP discrimination ratio as that in clean buffers. Therefore, this ultrasensitive SNP biosensor appears to be well-suited for potential diagnostic applications of genetic diseases.

Keywords: DNA detection, polymer beads, signal amplification, single nucleotide polymorphisms

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Authors: Priyal Chikhaliwala, Sudeshna Chandra

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Liver cancer is one of the most common malignant tumors with poor prognosis. This is because liver cancer does not exhibit any symptoms in early stage of disease. Increased serum level of AFP is clinically considered as a diagnostic marker for liver malignancy. The present diagnostic modalities include various types of immunoassays, radiological studies, and biopsy. However, these tests undergo slow response times, require significant sample volumes, achieve limited sensitivity and ultimately become expensive and burdensome to patients. Considering all these aspects, electrochemical biosensors based on dendrimer-magnetic nanoparticles (MNPs) was designed. Dendrimers are novel nano-sized, three-dimensional molecules with monodispersed structures. Poly-amidoamine (PAMAM) dendrimers with eight –NH₂ groups using ethylenediamine as a core molecule were synthesized using Michael addition reaction. Dendrimers provide added the advantage of not only stabilizing Fe₃O₄ NPs but also displays capability of performing multiple electron redox events and binding multiple biological ligands to its dendritic end-surface. Fe₃O₄ NPs due to its superparamagnetic behavior can be exploited for magneto-separation process. Fe₃O₄ NPs were stabilized with PAMAM dendrimer by in situ co-precipitation method. The surface coating was examined by FT-IR, XRD, VSM, and TGA analysis. Electrochemical behavior and kinetic studies were evaluated using CV which revealed that the dendrimer-Fe₃O₄ NPs can be looked upon as electrochemically active materials. Electrochemical immunosensor was designed by immobilizing anti-AFP onto dendrimer-MNPs by gluteraldehyde conjugation reaction. The bioconjugates were then incubated with AFP antigen. The immunosensor was characterized electrochemically indicating successful immuno-binding events. The binding events were also further studied using magnetic particle imaging (MPI) which is a novel imaging modality in which Fe₃O₄ NPs are used as tracer molecules with positive contrast. Multicolor MPI was able to clearly localize AFP antigen and antibody and its binding successfully. Results demonstrate immense potential in terms of biosensing and enabling MPI of AFP in clinical diagnosis.

Keywords: alpha-fetoprotein, dendrimers, electrochemical biosensors, magnetic nanoparticles

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Authors: Siti Nur Syazni Mohd Zuki, Tan Ling Ling, Nina Suhaity Azmi, Chong Kwok Feng, Lee Yook Heng

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Nitrite (NO2-) ion is used prevalently as a preservative in processed meat. Elevated levels of nitrite also found in edible bird’s nests (EBNs). Consumption of NO2- ion at levels above the health-based risk may cause cancer in humans. Spectrophotometric Griess test is the simplest established standard method for NO2- ion detection, however, it requires careful control of pH of each reaction step and susceptible to strong oxidants and dyeing interferences. Other traditional methods rely on the use of laboratory-scale instruments such as GC-MS, HPLC and ion chromatography, which cannot give real-time response. Therefore, it is of significant need for devices capable of measuring nitrite concentration in-situ, rapidly and without reagents, sample pretreatment or extraction step. Herein, we constructed a microspheres-based microbial optode for visual quantitation of NO2- ion. Raoutella planticola, the bacterium expressing NAD(P)H nitrite reductase (NiR) enzyme has been successfully extracted by microbial technique from EBN collected from local birdhouse. The whole cells and the lipophilic Nile Blue chromoionophore were physically absorbed on the photocurable poly(n-butyl acrylate-N-acryloxysuccinimide) [poly (nBA-NAS)] microspheres, whilst the reduced coenzyme NAD(P)H was covalently immobilized on the succinimide-functionalized acrylic microspheres to produce a reagentless biosensing system. Upon the NiR enzyme catalyzes the oxidation of NAD(P)H to NAD(P)+, NO2- ion is reduced to ammonium hydroxide, and that a colour change from blue to pink of the immobilized Nile Blue chromoionophore is perceived as a result of deprotonation reaction increasing the local pH in the microspheres membrane. The microspheres-based optosensor was optimized with a reflectance spectrophotometer at 639 nm and pH 8. The resulting microbial bio-optode membrane could quantify NO2- ion at 0.1 ppm and had a linear response up to 400 ppm. Due to the large surface area to mass ratio of the acrylic microspheres, it allows efficient solid state diffusional mass transfer of the substrate to the bio-recognition phase, and achieve the steady state response as fast as 5 min. The proposed optical microbial biosensor requires no sample pre-treatment step and possesses high stability as the whole cell biocatalyst provides protection to the enzymes from interfering substances, hence it is suitable for measurements in contaminated samples.

Keywords: acrylic microspheres, microbial bio-optode, nitrite ion, reflectometric

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Authors: Srinivas Bathini, Duraichelvan Raju, Simona Badilescu, Muthukumaran Packirisamy

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A bio-sensing method, based on the plasmonic property of gold nano-islands, has been developed for detection of exosomes in a clinical setting. The position of the gold plasmon band in the UV-Visible spectrum depends on the size and shape of gold nanoparticles as well as on the surrounding environment. By adsorbing various chemical entities, or binding them, the gold plasmon band will shift toward longer wavelengths and the shift is proportional to the concentration. Exosomes transport cargoes of molecules and genetic materials to proximal and distal cells. Presently, the standard method for their isolation and quantification from body fluids is by ultracentrifugation, not a practical method to be implemented in a clinical setting. Thus, a versatile and cutting-edge platform is required to selectively detect and isolate exosomes for further analysis at clinical level. The new sensing protocol, instead of antibodies, makes use of a specially synthesized polypeptide (Vn96), to capture and quantify the exosomes from different media, by binding the heat shock proteins from exosomes. The protocol has been established and optimized by using a glass substrate, in order to facilitate the next stage, namely the transfer of the protocol to a microfluidic environment. After each step of the protocol, the UV-Vis spectrum was recorded and the position of gold Localized Surface Plasmon Resonance (LSPR) band was measured. The sensing process was modelled, taking into account the characteristics of the nano-island structure, prepared by thermal convection and annealing. The optimal molar ratios of the most important chemical entities, involved in the detection of exosomes were calculated as well. Indeed, it was found that the results of the sensing process depend on the two major steps: the molar ratios of streptavidin to biotin-PEG-Vn96 and, the final step, the capture of exosomes by the biotin-PEG-Vn96 complex. The microfluidic device designed for sensing of exosomes consists of a glass substrate, sealed by a PDMS layer that contains the channel and a collecting chamber. In the device, the solutions of linker, cross-linker, etc., are pumped over the gold nano-islands and an Ocean Optics spectrometer is used to measure the position of the Au plasmon band at each step of the sensing. The experiments have shown that the shift of the Au LSPR band is proportional to the concentration of exosomes and, thereby, exosomes can be accurately quantified. An important advantage of the method is the ability to discriminate between exosomes having different origins.

Keywords: exosomes, gold nano-islands, microfluidics, plasmonic biosensing

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Authors: Neha Batra Bali, Monika Tomar, Vinay Gupta

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A reagentless biosensor for detection of urea based on ZnO-CuO composite thin film is presented in following work. Biosensors have immense potential for varied applications ranging from environmental to clinical testing, health care, and cell analysis. Immense growth in the field of biosensors is due to the huge requirement in today’s world to develop techniques which are both cost effective and accurate for prevention of disease manifestation. The human body comprises of numerous biomolecules which in their optimum levels are essential for functioning. However mismanaged levels of these biomolecules result in major health issues. Urea is one of the key biomolecules of interest. Its estimation is of paramount significance not only for healthcare sector but also from environmental perspectives. If level of urea in human blood/serum is abnormal, i.e., above or below physiological range (15-40mg/dl)), it may lead to diseases like renal failure, hepatic failure, nephritic syndrome, cachexia, urinary tract obstruction, dehydration, shock, burns and gastrointestinal, etc. Various metal nanoparticles, conducting polymer, metal oxide thin films, etc. have been exploited to act as matrix to immobilize urease to fabricate urea biosensor. Amongst them, Zinc Oxide (ZnO), a semiconductor metal oxide with a wide band gap is of immense interest as an efficient matrix in biosensors by virtue of its natural abundance, biocompatibility, good electron communication feature and high isoelectric point (9.5). In spite of being such an attractive candidate, ZnO does not possess a redox couple of its own which necessitates the use of electroactive mediators for electron transfer between the enzyme and the electrode, thereby causing hindrance in realization of integrated and implantable biosensor. In the present work, an effort has been made to fabricate a matrix based on ZnO-CuO composite prepared by pulsed laser deposition (PLD) technique in order to incorporate redox properties in ZnO matrix and to utilize the same for reagentless biosensing applications. The prepared bioelectrode Urs/(ZnO-CuO)/ITO/glass exhibits high sensitivity (70µAmM⁻¹cm⁻²) for detection of urea (5-200 mg/dl) with high stability (shelf life ˃ 10 weeks) and good selectivity (interference ˂ 4%). The enhanced sensing response obtained for composite matrix is attributed to the efficient electron exchange between ZnO-CuO matrix and immobilized enzymes, and subsequently fast transfer of generated electrons to the electrode via matrix. The response is encouraging for fabricating reagentless urea biosensor based on ZnO-CuO matrix.

Keywords: biosensor, reagentless, urea, ZnO-CuO composite

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Authors: Andreas Aceranti, Riccardo Dossena, Marco Colorato, Simonetta Vernocchi

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By the term “limbic resonance,” we usually refer to a state of deep connection, both emotional and physiological, between people who, when in resonance, find their limbic systems in tune with one another. Limbic resonance is not only about sharing emotions but also physiological states. In fact, people in such resonance can influence each other’s heart rate, blood pressure, and breathing. Limbic resonance is fundamental for human beings to connect and create deep bonds among a certain group. It is fundamental for our social skills. A relationship between gifted and resonant subjects is perceived as feeling safe, living the relation like an isle of serenity where it is possible to recharge, to communicate without words, to understand each others without giving explanations, to strengthen the balance of each member of the group. Within the circle, self-esteem is consolidated and makes it stronger to face what is outside, others, and reality. The idea that gifted people who are together may be unfit for the world does not correspond to the truth. The circle made up of people with high cognitive potential characterized by a limbic resonance is, in general, experienced as a solid platform from which you can safely move away and where you can return to recover strength. We studied 8 adults (between 21 and 47 years old). All of them with IQ higher than 130. We monitored their brain waves frequency (alpha, beta, theta, gamma, delta) by means of biosensing tracker along with their physiological states (heart beat frequency, blood pressure, breathing frequency, pO2, pCO2) and some blood works only (5-HT, dopamine, catecholamines, cortisol). The subjects of the study were asked to adhere to a protocol involving bonding activities (such as team building activities), role plays, meditation sessions, and group therapy. All these activities were carried out together. We observed that after about 4 months of activities, their brain waves frequencies tended to tune quicker and quicker. After 9 months, the bond among them was so important that they could “sense” each other inner states and sometimes also guess each others’ thoughts. According to our findings, it may be hypothesized that large synchronized outbursts of cortex neurons produces not only brain waves but also electromagnetic fields that may be able to influence the cortical neurons’ activity of other people’s brain by inducing action potentials in large groups of neurons and this is reasonably conceivable to be able to transmit information such as different emotions and cognition cues to the other’s brain. We also believe that upcoming research should focus on clarifying the role of brain magnetic particles in brain-to-brain communication. We also believe that further investigations should be carried out on the presence and role of cryptochromes to evaluate their potential roles in direct brain-to-brain communication.

Keywords: limbic resonance, psychotherapy, brain waves, emotion regulation, giftedness

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Authors: Mitali Saha, Soma Das

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The fabrication of nanoscale materials for use in chemical sensing, biosensing and biological analyses has proven a promising avenue in the last few years. Cholesterol has aroused considerable interest in recent years on account of its being an important parameter in clinical diagnosis. There is a strong positive correlation between high serum cholesterol level and arteriosclerosis, hypertension, and myocardial infarction. Enzyme-based electrochemical biosensors have shown high selectivity and excellent sensitivity, but the enzyme is easily denatured during its immobilization procedure and its activity is also affected by temperature, pH, and toxic chemicals. Besides, the reproducibility of enzyme-based sensors is not very good which further restrict the application of cholesterol biosensor. It has been demonstrated that carbon nanotubes could promote electron transfer with various redox active proteins, ranging from cytochrome c to glucose oxidase with a deeply embedded redox center. In continuation of our earlier work on the synthesis and applications of carbon and metal based nanoparticles, we have reported here the synthesis of carbon nanotubes (CCNT) by burning coconut oil under insufficient flow of air using an oil lamp. The soot was collected from the top portion of the flame, where the temperature was around 6500C which was purified, functionalized and then characterized by SEM, p-XRD and Raman spectroscopy. The SEM micrographs showed the formation of tubular structure of CCNT having diameter below 100 nm. The XRD pattern indicated the presence of two predominant peaks at 25.20 and 43.80, which corresponded to (002) and (100) planes of CCNT respectively. The Raman spectrum (514 nm excitation) showed the presence of 1600 cm-1 (G-band) related to the vibration of sp2-bonded carbon and at 1350 cm-1 (D-band) responsible for the vibrations of sp3-bonded carbon. A nonenzymatic cholesterol biosensor was then fabricated on an insulating Teflon material containing three silver wires at the surface, covered by CCNT, obtained from coconut oil. Here, CCNTs worked as working as well as counter electrodes whereas reference electrode and electric contacts were made of silver. The dimensions of the electrode was 3.5 cm×1.0 cm×0.5 cm (length× width × height) and it is ideal for working with 50 µL volume like the standard screen printed electrodes. The voltammetric behavior of cholesterol at CCNT electrode was investigated by cyclic voltammeter and differential pulse voltammeter using 0.001 M H2SO4 as electrolyte. The influence of the experimental parameters on the peak currents of cholesterol like pH, accumulation time, and scan rates were optimized. Under optimum conditions, the peak current was found to be linear in the cholesterol concentration range from 1 µM to 50 µM with a sensitivity of ~15.31 μAμM−1cm−2 with lower detection limit of 0.017 µM and response time of about 6s. The long-term storage stability of the sensor was tested for 30 days and the current response was found to be ~85% of its initial response after 30 days.

Keywords: coconut oil, CCNT, cholesterol, biosensor

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Authors: Moustafa Osman Mohammed

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This paper introduces topological order in descried social systems starting with the original concept of autopoiesis by biologists and scientists, including the modification of general systems based on socialized medicine. Topological order is important in describing the physical systems for exploiting optical systems and improving photonic devices. The stats of topological order have some interesting properties of topological degeneracy and fractional statistics that reveal the entanglement origin of topological order, etc. Topological ideas in photonics form exciting developments in solid-state materials, that being; insulating in the bulk, conducting electricity on their surface without dissipation or back-scattering, even in the presence of large impurities. A specific type of autopoiesis system is interrelated to the main categories amongst existing groups of the ecological phenomena interaction social and medical sciences. The hypothesis, nevertheless, has a nonlinear interaction with its natural environment 'interactional cycle' for exchange photon energy with molecules without changes in topology. The engineering topology of a biosensor is based on the excitation boundary of surface electromagnetic waves in photonic band gap multilayer films. The device operation is similar to surface Plasmonic biosensors in which a photonic band gap film replaces metal film as the medium when surface electromagnetic waves are excited. The use of photonic band gap film offers sharper surface wave resonance leading to the potential of greatly enhanced sensitivity. So, the properties of the photonic band gap material are engineered to operate a sensor at any wavelength and conduct a surface wave resonance that ranges up to 470 nm. The wavelength is not generally accessible with surface Plasmon sensing. Lastly, the photonic band gap films have robust mechanical functions that offer new substrates for surface chemistry to understand the molecular design structure and create sensing chips surface with different concentrations of DNA sequences in the solution to observe and track the surface mode resonance under the influences of processes that take place in the spectroscopic environment. These processes led to the development of several advanced analytical technologies: which are; automated, real-time, reliable, reproducible, and cost-effective. This results in faster and more accurate monitoring and detection of biomolecules on refractive index sensing, antibody-antigen reactions with a DNA or protein binding. Ultimately, the controversial aspect of molecular frictional properties is adjusted to each other in order to form unique spatial structure and dynamics of biological molecules for providing the environment mutual contribution in investigation of changes due to the pathogenic archival architecture of cell clusters.

Keywords: autopoiesis, photonics systems, quantum topology, molecular structure, biosensing

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Authors: Nicholas Mavrogiannis, Francesca Crivellari, Zachary Gagnon

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Development of low-cost, rapid, sensitive and portable biosensing systems are important for the detection and prevention of disease in developing countries, biowarfare/antiterrorism applications, environmental monitoring, point-of-care diagnostic testing and for basic biological research. Currently, the most established commercially available and widespread assays for portable point of care detection and disease testing are paper-based dipstick and lateral flow test strips. These paper-based devices are often small, cheap and simple to operate. The last three decades in particular have seen an emergence in these assays in diagnostic settings for detection of pregnancy, HIV/AIDS, blood glucose, Influenza, urinary protein, cardiovascular disease, respiratory infections and blood chemistries. Such assays are widely available largely because they are inexpensive, lightweight, and portable, are simple to operate, and a few platforms are capable of multiplexed detection for a small number of sample targets. However, there is a critical need for sensitive, quantitative and multiplexed detection capabilities for point-of-care diagnostics and for the detection and prevention of disease in the developing world that cannot be satisfied by current state-of-the-art paper-based assays. For example, applications including the detection of cardiac and cancer biomarkers and biothreat applications require sensitive multiplexed detection of analytes in the nM and pM range, and cannot currently be satisfied with current inexpensive portable platforms due to their lack of sensitivity, quantitative capabilities and often unreliable performance. In this talk, inexpensive label-free biomolecular detection at liquid interfaces using a newly discovered electrokinetic phenomenon known as fluidic dielectrophoresis (fDEP) is demonstrated. The electrokinetic approach involves exploiting the electrical mismatches between two aqueous liquid streams forced to flow side-by-side in a microfluidic T-channel. In this system, one fluid stream is engineered to have a higher conductivity relative to its neighbor which has a higher permittivity. When a “low” frequency (< 1 MHz) alternating current (AC) electrical field is applied normal to this fluidic electrical interface the fluid stream with high conductivity displaces into the low conductive stream. Conversely, when a “high” frequency (20MHz) AC electric field is applied, the high permittivity stream deflects across the microfluidic channel. There is, however, a critical frequency sensitive to the electrical differences between each fluid phase – the fDEP crossover frequency – between these two events where no fluid deflection is observed, and the interface remains fixed when exposed to an external field. To perform biomolecular detection, two streams flow side-by-side in a microfluidic T-channel: one fluid stream with an analyte of choice and an adjacent stream with a specific receptor to the chosen target. The two fluid streams merge and the fDEP crossover frequency is measured at different axial positions down the resulting liquid

Keywords: biodetection, fluidic dielectrophoresis, interfacial polarization, liquid interface

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Authors: Alena Semeradtova, Marcel Stofik, Lucie Mareckova, Petr Maly, Ondrej Stanek, Jan Maly

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Recently, novel strategies based on so-called molecular evolution were shown to be effective for the production of various peptide ligand libraries with high affinities to molecular targets of interest comparable or even better than monoclonal antibodies. The major advantage of these peptide scaffolds is mainly their prevailing low molecular weight and simple structure. This study describes a new high-affinity binding molecules based immunesensor using a simple optical system for human serum albumin (HSA) detection as a model molecule. We present a comparison of two variants of recombinant binders based on albumin binding domain of the protein G (ABD) performed on micropatterned glass chip. Binding domains may be tailored to any specific target of interest by molecular evolution. Micropatterened glass chips were prepared using UV-photolithography on chromium sputtered glasses. Glass surface was modified by (3-aminopropyl)trietoxysilane and biotin-PEG-acid using EDC/NHS chemistry. Two variants of high-affinity binding molecules were used to detect target molecule. Firstly, a variant is based on ABD domain fused with TolA chain. This molecule is in vivo biotinylated and each molecule contains one molecule of biotin and one ABD domain. Secondly, the variant is ABD domain based on streptavidin molecule and contains four gaps for biotin and four ABD domains. These high-affinity molecules were immobilized to the chip surface via biotin-streptavidin chemistry. To eliminate nonspecific binding 1% bovine serum albumin (BSA) or 6% fetal bovine serum (FBS) were used in every step. For both variants range of measured concentrations of fluorescently labelled HSA was 0 – 30 µg/ml. As a control, we performed a simultaneous assay without high-affinity binding molecules. Fluorescent signal was measured using inverse fluorescent microscope Olympus IX 70 with COOL LED pE 4000 as a light source, related filters, and camera Retiga 2000R as a detector. The fluorescent signal from non-modified areas was substracted from the signal of the fluorescent areas. Results were presented in graphs showing the dependence of measured grayscale value on the log-scale of HSA concentration. For the TolA variant the limit of detection (LOD) of the optical immunosensor proposed in this study is calculated to be 0,20 µg/ml for HSA detection in 1% BSA and 0,24 µg/ml in 6% FBS. In the case of streptavidin-based molecule, it was 0,04 µg/ml and 0,07 µg/ml respectively. The dynamical range of the immunosensor was possible to estimate just in the case of TolA variant and it was calculated to be 0,49 – 3,75 µg/ml and 0,73-1,88 µg/ml respectively. In the case of the streptavidin-based the variant we didn´t reach the surface saturation even with the 480 ug/ml concentration and the upper value of dynamical range was not estimated. Lower value was calculated to be 0,14 µg/ml and 0,17 µg/ml respectively. Based on the obtained results, it´s clear that both variants are useful for creating the bio-recognizing layer on immunosensors. For this particular system, it is obvious that the variant based on streptavidin molecule is more useful for biosensing on glass planar surfaces. Immunosensors based on this variant would exhibit better limit of detection and wide dynamical range.

Keywords: high affinity binding molecules, human serum albumin, optical immunosensor, protein G, UV-photolitography

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Authors: Francesca Crivellari, Nicholas Mavrogiannis, Zachary Gagnon

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Portable diagnostic methods have become increasingly important for a number of different purposes: point-of-care screening in developing nations, environmental contamination studies, bio/chemical warfare agent detection, and end-user use for commercial health monitoring. The cheapest and most portable methods currently available are paper-based – lateral flow and dipstick methods are widely available in drug stores for use in pregnancy detection and blood glucose monitoring. These tests are successful because they are cheap to produce, easy to use, and require minimally invasive sampling. While adequate for their intended uses, in the realm of blood-borne pathogens and numerous cancers, these paper-based methods become unreliable, as they lack the nM/pM sensitivity currently achieved by clinical diagnostic methods. Clinical diagnostics, however, utilize techniques involving surface plasmon resonance (SPR) and enzyme-linked immunosorbent assays (ELISAs), which are expensive and unfeasible in terms of portability. To develop a better, competitive biosensor, we must reduce the cost of one, or increase the sensitivity of the other. Electric fields are commonly utilized in microfluidic devices to manipulate particles, biomolecules, and cells. Applications in this area, however, are primarily limited to interfaces formed between immiscible interfaces. Miscible, liquid-liquid interfaces are common in microfluidic devices, and are easily reproduced with simple geometries. Here, we demonstrate the use of electrical fields at liquid-liquid electrical interfaces, known as fluidic dielectrophoresis, (fDEP) for biodetection in a microfluidic device. In this work, we apply an AC electric field across concurrent laminar streams with differing conductivities and permittivities to polarize the interface and induce a discernible, near-immediate, frequency-dependent interfacial tilt. We design this aqueous electrical interface, which becomes the biosensing “substrate,” to be intelligent – it “moves” only when a target of interest is present. This motion requires neither labels nor expensive electrical equipment, so the biosensor is inexpensive and portable, yet still capable of sensitive detection. Nanoparticles, due to their high surface-area-to-volume ratio, are often incorporated to enhance detection capabilities of schemes like SPR and fluorimetric assays. Most studies currently investigate binding at an immobilized solid-liquid or solid-gas interface, where particles are adsorbed onto a planar surface, functionalized with a receptor to create a reactive substrate, and subsequently flushed with a fluid or gas with the relevant analyte. These typically involve many preparation and rinsing steps, and are susceptible to surface fouling. Our microfluidic device is continuously flowing and renewing the “substrate,” and is thus not subject to fouling. In this work, we demonstrate the ability to electrokinetically detect biomolecules binding to functionalized nanoparticles at liquid-liquid interfaces using fDEP. In biotin-streptavidin experiments, we report binding detection limits on the order of 1-10 pM, without amplifying signals or concentrating samples. We also demonstrate the ability to detect this interfacial motion, and thus the presence of binding, using impedance spectroscopy, allowing this scheme to become non-optical, in addition to being label-free.

Keywords: biodetection, dielectrophoresis, microfluidics, nanoparticles

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Authors: Andreea Campu, Ilinica Muresan, Simona Cainap, Simion Astilean, Monica Focsan

Abstract:

Acute myocardial infarction (AMI) is the most severe cardiovascular disease, which has threatened human lives for decades, thus a continuous interest is directed towards the detection of cardiac biomarkers such as cardiac troponin I (cTnI) in order to predict risk and, implicitly, fulfill the early diagnosis requirements in AMI settings. Microfluidics is a major technology involved in the development of efficient sensing devices with real-time fast responses and on-site applicability. Microfluidic devices have gathered a lot of attention recently due to their advantageous features such as high sensitivity and specificity, miniaturization and portability, ease-of-use, low-cost, facile fabrication, and reduced sample manipulation. The integration of gold nanoparticles into the structure of microfluidic sensors has led to the development of highly effective detection systems, considering the unique properties of the metallic nanostructures, specifically the Localized Surface Plasmon Resonance (LSPR), which makes them highly sensitive to their microenvironment. In this scientific context, herein, we propose the implementation of a novel detection device, which successfully combines the efficiency of gold bipyramids (AuBPs) as signal transducers and thermal generators with the sample-driven advantages of the microfluidic channels into a miniaturized, portable, low-cost, specific, and sensitive test for the dual LSPR-thermographic cTnI detection. Specifically, AuBPs with longitudinal LSPR response at 830 nm were chemically synthesized using the seed-mediated growth approach and characterized in terms of optical and morphological properties. Further, the colloidal AuBPs were deposited onto pre-treated silanized glass substrates thus, a uniform nanoparticle coverage of the substrate was obtained and confirmed by extinction measurements showing a 43 nm blue-shift of the LSPR response as a consequence of the refractive index change. The as-obtained plasmonic substrate was then integrated into a microfluidic “Y”-shaped polydimethylsiloxane (PDMS) channel, fabricated using a Laser Cutter system. Both plasmonic and microfluidic elements were plasma treated in order to achieve a permanent bond. The as-developed microfluidic plasmonic chip was further coupled to an automated syringe pump system. The proposed biosensing protocol implicates the successive injection inside the microfluidic channel as follows: p-aminothiophenol and glutaraldehyde, to achieve a covalent bond between the metallic surface and cTnI antibody, anti-cTnI, as a recognition element, and target cTnI biomarker. The successful functionalization and capture of cTnI was monitored by LSPR detection thus, after each step, a red-shift of the optical response was recorded. Furthermore, as an innovative detection technique, thermal determinations were made after each injection by exposing the microfluidic plasmonic chip to 785 nm laser excitation, considering that the AuBPs exhibit high light-to-heat conversion performances. By the analysis of the thermographic images, thermal curves were obtained, showing a decrease in the thermal efficiency after the anti-cTnI-cTnI reaction was realized. Thus, we developed a microfluidic plasmonic chip able to operate as both LSPR and thermal sensor for the detection of the cardiac troponin I biomarker, leading thus to the progress of diagnostic devices.

Keywords: gold nanobipyramids, microfluidic device, localized surface plasmon resonance detection, thermographic detection

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