Developing Novel Functional Laser-Induced Carbon Nanofibers for Miniaturized Electroanalytical Biosensors

Abstract

Electrochemical sensors are ideal candidates for point-of-care diagnostics due to their low cost, sensitivity, and direct data readout. Nowadays, enzyme-based sensors dominate the electrochemical diagnostics market due to their outstanding selectivity. However, there are some drawbacks related to the use of enzyme like stability issues or high manufacturing costs. This thesis highlights the need for enzyme-free alternatives and provides a comprehensive overview of non-enzymatic sensors and their development. The definition, principles, and typical detection mechanisms of electrochemical sensing are explained. Various conventional strategies for fabricating enzyme-free electrochemical sensors, such as drop-casting, dip-coating, electro- and electroless deposition, screen/inkjet/3D-/roll-to-roll printing, and the emerging technology of laser scribing, are introduced and compared in terms of cost, complexity, mass production capability, and their individual pros and cons. Additionally, considerations regarding selectivity, sensitivity, and biofouling when applying these sensors to different matrices, such as clinical samples (blood, dermal interstitial fluid, sweat, saliva, urine, breath) and non-clinical samples (water from rivers, lakes, seas, food and beverages, cell culture media), are presented. Other challenges and potential solutions related to electrode fouling, measurements under physiological conditions, biocompatibility, long-term stability, storage, practical issues, and efficiency are also discussed. In this thesis, a non-enzymatic electrochemical sensor based on laser-induced carbon nanofibers (LCNFs) for detecting hydrogen peroxide was developed. The working electrode was laser-scribed on polyimide nanofibers produced by electrospinning. These fibers contained either one or a mixture of both metal salts Pt(II)- and Ni(II)-acetylacetonate, which were converted to nanoparticles embedded within the LCNF during the laser treatment. The resulting nanoparticles consisted either of Pt- or Ni- metals and their oxides, or Pt-Ni-alloyed metal and its oxide in the case of a mixture. In the measurement setup, a Pt-wire and an Ag/AgCl electrode were used as the counter and reference electrodes, respectively, to investigate hydrogen peroxide detection. LCNFs with various compositions of the metal salts were tested towards the catalytic behavior to hydrogen peroxide and their selectivity. The best sensitivity (with a limit of detection (LOD) of 1.4 ± 0.4 μM) in an un-stirred approach was achieved by oxidizing hydrogen peroxide on a pure Pt-LCNF, but this led to unsatisfactory selectivity. Strategies such as increasing the metal content or using polymer coatings were applied to improve selectivity. Drop-coating the electrode with nylon also improved the recovery of hydrogen peroxide spiked in real matrices (undiluted and diluted human serum). Selectivity was further improved by reducing the measurement potential and switching from oxidation to reduction of hydrogen peroxide, although this came at the cost of sensitivity. Additionally, strategies to attach polyoxometalates as alternative catalysts onto the LCNF surface were explored but did not result in sensitive hydrogen peroxide detection. A second project in this thesis focused on the non-enzymatic electrochemical detection of glucose under physiological conditions. It is well-known that glucose oxidation is catalyzed on Ni-surfaces at an alkaline pH. Therefore, the Pt/Ni-alloyed LCNF described in the previous project was used to efficiently generate a high local pH by an electrochemical pretreatment (-0.9 V for 20 s) on the Pt-sites of the LCNF, enabling glucose measurement on the Ni-sites of the alloy in a consecutive cyclovoltammetry measurement, even though the initial pH of the solution was 7.4. To enable long-term measurements, an electrochemical treatment after each measurement was introduced to clean the catalyst surface. After optimizing (i) the pretreatment, (ii) the catalyst cleaning, (iii) the measurement conditions, and (iv) the data treatment, glucose could be selectively detected in physiologically relevant concentrations with a LOD of 0.3 ± 0.1 mM. Furthermore, the aging of the electrode was investigated, where hydrophobicity is increasing with time, leading to a reduced contact between electrode and solution. To resolve this, a simple Tween-20 incubation was applied prior to measurement. Finally, the electrode performance was not affected after sterilization, and a recovery of 95 ± 10% in spiked, diluted human serum was achieved. In the last project, pure Ni-LCNF were laser-scribed as a 3-electrode system (LCNF was used as the working, counter, and reference electrode) and used to detect glucose in simulated breath. Glucose solutions with concentrations similar to those present in lung fluids were nebulized, captured on the porous LCNFs, and electrochemically measured in NaOH solution. With optimized manufacturing, glucose-capturing, and measurement parameters, glucose was detected selectively using cyclovoltammetry and a ratiometric data readout. To simulate variations in lung fluid concentration (reflecting blood sugar level variations), various glucose concentrations of the nebulized solutions were captured, measured, and could be distinguished from each other