Advanced Biosensing towards Real-Time Imaging of Protein Secretion from Single Cells

Protein secretion of cells plays a vital role in intercellular communication. The abnormality and dysfunction of cellular protein secretion are associated with various physiological disorders, such as malignant proliferation of cells, aberrant immune function, and bone marrow failure. The heterogeneity of protein secretion exists not only between varying populations of cells, but also in the same phenotype of cells. Therefore, characterization of protein secretion from single cell contributes not only to the understanding of intercellular communication in immune effector, carcinogenesis and metastasis, but also to the development and improvement of diagnosis and therapy of relative diseases. In spite of abundant highly sensitive methods that have been developed for the detection of secreted proteins, majority of them fall short in providing sufficient spatial and temporal resolution for comprehensive profiling of protein secretion from single cells. The real-time imaging techniques allow rapid acquisition and manipulation of analyte information on a 2D plane, providing high spatiotemporal resolution. Here, we summarize recent advances in real-time imaging of secretory proteins from single cell, including label-free and labelling techniques, shedding light on the development of simple yet powerful methodology for real-time imaging of single-cell protein secretion

Mass Transfer in Amperometric Biosensors Based on Nanocomposite Thin Films of Redox Polymers and Oxidoreductases

Mass transfer in nanocomposite hydrogel thin films consisting of alternating layers of an organometallic redox polymer (RP) and oxidoreductase enzymes was investigated. Multilayer nanostructures were fabricated on gold surfaces by the deposition of an anionic self-assembled monolayer of 11-mercaptoundecanoic acid, followed by the electrostatic binding of a cationic redox polymer, poly[vinylpyridine Os(bis-bipyridine)2Clco-allylamine], and an anionic oxidoreductase. Surface plasmon resonance spectroscopy, Fourier transform infrared external reflection spectroscopy (FTIR-ERS), ellipsometry and electrochemistry were employed to characterize the assembly of these nanocomposite films. Simultaneous SPR/electrochemistry enabled real time observation of the assembly of sensing components, changes in film structure with electrode potential, and the immediate, in situ electrochemical verification of substrate-dependent current upon the addition of enzyme to the multilayer structure. SPR and FTIR-ERS studies also showed no desorption of polymer or enzyme from the nanocomposite structure when stored in aqueous environment occurred over the period of three weeks, suggesting that decreasing in substrate sensitivity were due to loss of enzymatic activity rather than loss of film compounds from the nanostructure

Enzymatic Logic Gates with Noise-Reducing Sigmoid Response

Biochemical computing is an emerging field of unconventional computing that attempts to process information with biomolecules and biological objects using digital logic. In this work we survey filtering in general, in biochemical computing, and summarize the experimental realization of an AND logic gate with sigmoid response in one of the inputs. The logic gate is realized with electrode-immobilized glucose-6-phosphate dehydrogenase enzyme that catalyzes a reaction corresponding to the Boolean AND functions. A kinetic model is also developed and used to evaluate the extent to which the performance of the experimentally realized logic gate is close to optimal.Comment: 14 pages, 2 figures, PD

Enzymatic AND-Gate Based on Electrode-Immobilized Glucose-6-Phosphate Dehydrogenase: Towards Digital Biosensors and Biochemical Logic Systems with Low Noise

Electrode-immobilized glucose-6-phosphate dehydrogenase is used to catalyze an enzymatic reaction which carries out the AND logic gate. This logic function is considered here in the context of biocatalytic processes utilized for the biocomputing applications for "digital" (threshold) sensing/actuation. We outline the response functions desirable for such applications and report the first experimental realization of a sigmoid-shape response in one of the inputs. A kinetic model is developed and utilized to evaluate the extent to which the experimentally realized gate is close to optimal

Special Issue—Biosensors and Neuroscience: Is Biosensors Engineering Ready to Embrace Design Principles from Neuroscience?

In partnership with the Air Force Office of Scientific Research (AFOSR), the National Science Foundation’s (NSF) Emerging Frontiers and Multidisciplinary Activities (EFMA) office of the Directorate for Engineering (ENG) launched an Emerging Frontiers in Research and Innovation (EFRI) topic for the fiscal years FY22 and FY23 entitled “Brain-inspired Dynamics for Engineering Energy-Efficient Circuits and Artificial Intelligence” (BRAID) [...

Extended Linear Response for Bioanalytical Applications Using Multiple Enzymes

We develop a framework for optimizing a novel approach to extending the linear range of bioanalytical systems and biosensors by utilizing two enzymes with different kinetic responses to the input chemical as their substrate. Data for the flow injection amperometric system devised for detection of lysine based on the function of l-lysine-alpha-oxidase and lysine-2-monooxygenase are analyzed. Lysine is a homotropic substrate for the latter enzyme. We elucidate the mechanism for extending the linear response range and develop optimization techniques for future applications of such systems

Micropatterned nanocomposite hydrogels for biosensing applications

This paper describes the use of Au nanoparticle (NP)-containing hydrogel microstructures in the development of electrochemical enzyme-based biosensors. To fabricate biosensors, AuNPs were conjugated with glucose oxidase (GOX) or horseradish peroxidase (HRP) molecules and were dispersed in the prepolymer solution of poly(ethylene glycol) diacrylate (PEG-DA). Vinylferrocene (VF) was also added into the prepolymer solution in order to lower operating potential of the biosensor and to prevent oxidation of interfering substances. The prepolymer solution was photolithographically patterned in alignment with an array of Au electrodes fabricated on glass. As a result, electrode arrays became functionalized with AuNP/GOX- or AuNP/HRP-carrying hydrogel microstructures. Performance of the biosensors was characterized by impedance spectroscopy, chronoapmerometry and cyclic voltammetry. Impedance measurements revealed that inclusion of Au nanoparticles improved conductivity of PEG hydrogel by a factor of 5. Importantly, biosensors based on AuNP-GOX complex exhibited high sensitivity to glucose (100μAmM -1cm -2) in the linear range from 0.1 to 10mM. The detection limit was estimated to be 3.7×10- 7M at a signal-to-noise ratio of 3. Biosensors with immobilized AuNP/HPR had a linear response from 0.5 to 5.0μM of hydrogen peroxide with sensitivity of 1.4mAmM -1cm -2. The method for fabricating nanoparticle-carrying hydrogel microstructures described in this paper should be widely applicable in the development of robust and sensitive electrochemical biosensors. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim