Detection and monitoring of lactate and glucose levels in biological fluids and cell cultures are essential for understanding metabolic disorders. While electrochemical biosensors are commonly used, traditional enzymatic sensors face challenges related to stability, reproducibility, and cost. To address these limitations, we developed non-enzymatic sensors for lactate and glucose detection using nanostructured nickel oxide (NiO)–modified screen-printed carbon electrodes. The sensors were fabricated by drop-casting a NiO/Nafion/ethanol dispersion onto the working electrode, and their performance was evaluated using cyclic voltammetry and amperometry. Optimal sensitivity and linearity were achieved at a working potential of ∼0.5 V. The sensors exhibited linear responses for both lactate and glucose in the 0.1–5 mM range, with detection limits of 0.03 mM (lactate) and 0.025 mM (glucose), and sensitivities of 1.564 μA/mM (lactate) and 1.842 μA/mM (glucose) in 0.1 M NaOH–KCl electrolyte. To address glucose interference in lactate sensing, dual-sensing strategies were employed by varying Nafion concentration, applying differential potentials, or modifying the sensors with Prussian Blue to achieve selective detection. Validation against commercial lactate and glucose assay kits in cell culture medium showed good agreement, confirming the sensors’ accuracy. Finally, the sensor was integrated with a microfluidic chip, demonstrating its potential as a flow-through, enzyme-free metabolic sensor for future organ-on-a-chip applications.

Biology and medicine have seen groundbreaking discoveries, from ion channels to induced pluripotent stem cells, resulting in a paradigm shift. The advancements in physical sciences and engineering have always been pivotal in unlocking mysteries of biology and highlighting that the new frontiers lie in deepening our understanding at the single-cell and single-molecule levels. Applying different physical and engineering principles sheds new light on our understanding of complex biological systems at the single-cell and single-molecule level, enabling the development of various technologies such as single-molecule detection, organ-on-chip platforms, and organoids. The development of these technologies offers valuable insights into disease progression and personalized therapeutic strategies. The advancements in micro and nanofabrication propel the development of sensing platforms and biological devices that pave the way for novel solutions, ensuring the best of both worlds. This thesis aims to contribute to advancing the fields of single-molecule sensing and cell therapy by integrating biological discoveries and engineering advancements to develop novel engineering toolboxes. 

The first part of this thesis introduces and describes two approaches for single-molecule sensing and detection, specifically tunneling nanogaps and solid-state nanopore-based sensing platforms. The first work reports the custom measurement setup built during the project, which facilitates automated probing and testing arrays with hundreds of tunnel junctions in liquid with integrated microfluidics, current in the pA range, and at sampling rates up to 200 kHz. This setup highlights key electrical and microfluidic components and design choices to achieve a scalable measurement method, providing a platform for further studies and development in this field and enabling the potential for dynamic sensing. The second work in this thesis investigates the fabrication and electrical behavior of tunnel junctions in various gaseous and liquid media by feedback-controlled electromigration of microfabricated gold nanoconstrictions. This work maps the conductance stability and characteristics of the resulting tunnel junctions, highlighting various considerations and challenges in working with on-chip integrated tunnel junctions to guide future efforts. 

In the third work, we shift our focus to solid-state nanopores and demonstrate that the nanopores fabricated by controlled dielectric breakdown could be localized at the site of femtosecond laser exposure on a pristine silicon nitride membrane. We analyze the sensing potential of these nanopores by the translocation of double-stranded DNA through the pores. The fourth work uses the solid-state nanopore platform to detect and study the binding of Estrogen Receptor Alpha to the Estrogen Receptor Elements on the DNA. The work on tunnel junction and solid-state nanopore-based sensing modalities holds potential for further development in the field of single-(bio)molecule sensing.

The second part of this thesis presents a microfluidic chip platform that enables simple and fast reprogramming of somatic cells, such as fibroblasts, into induced pluripotent stem cells (iPSCs). These iPSCs can then be differentiated further into functional ectodermal cell types towards neural lineage, resulting in neural stem cells on the chip. Furthermore, using bulk-RNA sequencing, we observed that the microfluidic platform boosted commitment toward generating neural stem cells while reducing biological variability compared to a conventional well plate. Our method provides a simple platform with considerably reduced reagent requirements, cellular input, and manual labor, leading to substantial cost savings and holding potential for the highly controlled generation of clinical-grade iPSCs and differentiated cells for cellular therapeutics.

Banbrytande upptäckter inom biologi och medicin, från jonkanaler till inducerade pluripotenta stamceller, har resulterat i ett paradigmskifte. Framsteg inom fysik och ingenjörsvetenskap har varit avgörande för att avslöja biologins mysterier och belysa att framtidens nya avancemang ligger i att fördjupa vår förståelse på enskild cell- och molekylnivå. Genom att tillämpa olika fysikaliska och ingenjörsvetenskapliga principer får vi nya insikter gällande komplexa biologiska system på dessa nivåer, vilket möjliggör utvecklingen av olika teknologier såsom detektion av enskilda molekyler, organ-på-chip-plattformar och organoider. Utvecklingen av dessa teknologier ger värdefulla insikter för sjukdomsprogressioner och individanpassade terapeutiska strategier. Framstegen inom mikro- och nanofabrikation driver utvecklingen av sensorplattformar och biologiska enheter som banar väg för nya lösningar där det bästa från två världar förenas. Denna avhandling syftar till att bidra till utvecklingen av fälten för enskild molekyl-detektion och cellterapi genom att integrera biologiska upptäckter med ingenjörsvetenskapliga framsteg för att skapa nya teknologiska verktyg.

Den första delen av denna avhandling introducerar och beskriver två metoder för detektion av enskilda molekyler, specifikt tunnel-nanogap och fasta nanoporer som sensorplattformar. Den första studien rapporterar om den specialanpassade mätuppställning som byggdes under projektet och som möjliggör automatiserad avläsning och testning av arrays med hundratals tunnelövergångar i vätska med integrerad mikrofluidik, strömmar i pA-området och samplingsfrekvenser upp till 200 kHz. Systemet belyser viktiga elektriska och mikrofluidiska komponenter samt de designval som krävs för att uppnå en skalbar mätmetod, vilket skapar en plattform för vidare studier och utveckling inom detta fält och möjliggör potentialen för dynamisk detektion. Den andra studien i denna avhandling undersöker tillverkningen och den elektriska funktionen hos tunnelövergångar i olika gas- och vätskemedier genom återkopplingsstyrd elektromigration av mikrofabrikerade guldkonstriktioner. Detta arbete kartlägger ledningsstabiliteten och egenskaperna hos de resulterande tunnelövergångarna och belyser olika överväganden och utmaningar vid arbete med on-chip-integrerade tunnelövergångar för att vägleda framtida insatser.

I den tredje studien skiftar vi fokus till fasta nanoporer och demonstrerar att nanoporer, som skapats genom kontrollerad dielektrisk nedbrytning, kan lokaliseras till platsen för femtosekundslaserexponering på ett orört kiselnitridmembran. Vi analyserar sensorpotentialen hos dessa nanoporer genom translokation av dubbelsträngat DNA genom porerna. Den fjärde studien använder den fasta nanoporplattformen för att detektera och studera bindningen av östrogenreceptor alfa till östrogenreceptor-element på DNA. Arbetet med tunnelövergångar och fasta nanoporbaserade sensorplattformar har potential för vidare utveckling inom fältet för enskild-(bio)molekyl-detektion.

Den andra delen av denna avhandling presenterar en mikrofluidisk chip-plattform som möjliggör enkel och snabb omprogrammering av somatiska celler, såsom fibroblaster, till inducerade pluripotenta stamceller (iPSCs). Dessa iPSCs kan sedan vidare differentieras till funktionella ektodermala celltyper mot neural linje, vilket resulterar i neurala stamceller på chipet. Vidare observerade vi genom bulk-RNA-sekvensering att den mikrofluidiska plattformen främjade genererering av neurala stamceller samtidigt som den minskade biologisk variation jämfört med en konventionell brunnsplatta. Vår metod tillhandahåller en enkel plattform med avsevärt minskade reagenskrav, cellinsatser och manuellt arbete, vilket leder till betydande kostnadsbesparingar och har potential för högkontrollerad produktion av kliniskt godkända iPSCs och differentierade celler för cellulära terapier.

Controlled breakdown has emerged as an effective method for fabricating solid-state nanopores in thin suspended dielectric membranes for various biomolecular sensing applications. On an unpatterned membrane, the site of nanopore formation by controlled breakdown is random. Nanopore formation on a specific site on the membrane has previously been realized using local thinning of the membrane by lithographic processes or laser-assisted photothermal etching under immersion in an aqueous salt solution. However, these approaches require elaborate and expensive cleanroom-based lithography processes or involve intricate procedures using custom-made equipment. Here, we present a rapid cleanroom-free approach using single pulse femtosecond laser exposures of 50 nm thick silicon nitride membranes in air to localize the site of nanopore formation by subsequent controlled breakdown to an area less than 500 nm in diameter on the membrane. The precise positioning of the nanopores on the membrane could be produced both using laser exposure powers which caused significant thinning of the silicon nitride membrane (up to 60% of the original thickness locally), as well as at laser powers which caused no visible modification of the membrane at all. We show that nanopores made using our approach can work as single-molecule sensors by performing dsDNA translocation experiments. Due to the applicability of femtosecond laser processing to a wide range of membrane materials, we expect our approach to simplify the fabrication of localized nanopores by controlled breakdown in a variety of thin film material stacks, thereby enabling more sophisticated nanopore sensors.

Tunnel junctions have been suggested as high-throughput electronic single molecule sensors in liquids with several seminal experiments conducted using break junctions with reconfigurable gaps. For practical single molecule sensing applications, arrays of on-chip integrated fixed-gap tunnel junctions that can be built into compact systems are preferable. Fabricating nanogaps by electromigration is one of the most promising approaches to realize on-chip integrated tunnel junction sensors. However, the electrical behavior of fixed-gap tunnel junctions immersed in liquid media has not been systematically studied to date, and the formation of electromigrated nanogap tunnel junctions in liquid media has not yet been demonstrated. In this work, we perform a comparative study of the formation and electrical behavior of arrays of gold nanogap tunnel junctions made by feedback-controlled electromigration immersed in various liquid and gaseous media (deionized water, mesitylene, ethanol, nitrogen, and air). We demonstrate that tunnel junctions can be obtained from microfabricated gold nanoconstrictions inside liquid media. Electromigration of junctions in air produces the highest yield (61–67%), electromigration in deionized water and mesitylene results in a lower yield than in air (44–48%), whereas electromigration in ethanol fails to produce viable tunnel junctions due to interfering electrochemical processes. We map out the stability of the conductance characteristics of the resulting tunnel junctions and identify medium-specific operational conditions that have an impact on the yield of forming stable junctions. Furthermore, we highlight the unique challenges associated with working with arrays of large numbers of tunnel junctions in batches. Our findings will inform future efforts to build single molecule sensors using on-chip integrated tunnel junctions.

Tunnel junctions have long been used to immobilize and study the electronic transport properties of single molecules. The sensitivity of tunneling currents to entities in the tunneling gap has generated interest in developing electronic biosensors with single molecule resolution. Tunnel junctions can, for example, be used for sensing bound or unbound DNA, RNA, amino acids, and proteins in liquids. However, manufacturing technologies for on-chip integrated arrays of tunnel junction sensors are still in their infancy, and scalable measurement strategies that allow the measurement of large numbers of tunneling junctions are required to facilitate progress. Here, we describe an experimental setup to perform scalable, high-bandwidth (>10 kHz) measurements of low currents (pA–nA) in arrays of on-chip integrated tunnel junctions immersed in various liquid media. Leveraging a commercially available compact 100 kHz bandwidth low-current measurement instrument, we developed a custom two-terminal probe on which the amplifier is directly mounted to decrease parasitic probe capacitances to sub-pF levels. We also integrated a motorized three-axis stage, which could be powered down using software control, inside the Faraday cage of the setup. This enabled automated data acquisition on arrays of tunnel junctions without worsening the noise floor despite being inside the Faraday cage. A deliberately positioned air gap in the fluidic path ensured liquid perfusion to the chip from outside the Faraday cage without coupling in additional noise. We demonstrate the performance of our setup using rapid current switching observed in electromigrated gold tunnel junctions immersed in deionized water.

Organic mixed ionic-electronic conductors are promising materials for interfacing and monitoring biological systems, with the aim of overcoming current challenges based on the mismatch between biological materials and convectional inorganic conductors. The conjugated polymer/polyelectrolyte complex poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT/PSS) is, up to date, the most widely used polymer for in vitro or in vivo measurements in the field of organic bioelectronics. However, PEDOT/PSS organic electrochemical transistors (OECTs) are limited by depletion mode operation and lack chemical groups that enable synthetic modifications for biointerfacing. Recently introduced thiophene-based polymers with oligoether side chains can operate in accumulation mode, and their chemical structure can be tuned during synthesis, for example, by the introduction of hydroxylated side chains. Here, we introduce a new thiophene-based conjugated polymer, p(g42T-T)-8% OH, where 8% of the glycol side chains are functionalized with a hydroxyl group. We report for the first time the compatibility of conjugated polymers containing ethylene glycol side chains in direct contact with cells. The additional hydroxyl group allows covalent modification of the surface of polymer films, enabling fine-tuning of the surface interaction properties of p(g42T-T)-8% OH with biological materials, either hindering or promoting cell adhesion. We further use p(g42T-T)-8% OH to fabricate the OECTs and demonstrate for the first time the monitoring of epithelial barrier formation of Caco-2 cells in vitro using accumulation mode OECTs. The conjugated polymer p(g42T-T)-8% OH allows organic-electronic-based materials to be easily modified and optimized to interface and monitor biological systems.

Diluting organic semiconductors with a host insulating polymer is used to increase the electronic mobility in organic electronic devices, such as thin film transistors, while considerably reducing material costs. In contrast to organic electronics, bioelectronic devices such as the organic electrochemical transistor (OECT) rely on both electronic and ionic mobility for efficient operation, making it challenging to integrate hydrophobic polymers as the predominant blend component. This work shows that diluting the n-type conjugated polymer p(N-T) with high molecular weight polystyrene (10 KDa) leads to OECTs with over three times better mobility-volumetric capacitance product (µC*) with respect to the pristine p(N-T) (from 4.3 to 13.4 F V−1 cm−1 s−1) while drastically decreasing the amount of conjugated polymer (six times less). This improvement in µC* is due to a dramatic increase in electronic mobility by two orders of magnitude, from 0.059 to 1.3 cm2 V−1 s−1 for p(N-T):Polystyrene 10 KDa 1:6. Moreover, devices made with this polymer blend show better stability, retaining 77% of the initial drain current after 60 minutes operation in contrast to 12% for pristine p(N-T). These results open a new generation of low-cost organic mixed ionic-electronic conductors where the bulk of the film is made by a commodity polymer.

The clinical translation of induced pluripotent stem cells (iPSCs) holds great potential for personalized therapeutics. However, one of the main obstacles is that the current workflow to generate iPSCs is expensive, time-consuming, and requires standardization. A simplified and cost-effective microfluidic approach is presented for reprogramming fibroblasts into iPSCs and their subsequent differentiation into neural stem cells (NSCs). This method exploits microphysiological technology, providing a 100-fold reduction in reagents for reprogramming and a ninefold reduction in number of input cells. The iPSCs generated from microfluidic reprogramming of fibroblasts show upregulation of pluripotency markers and downregulation of fibroblast markers, on par with those reprogrammed in standard well-conditions. The NSCs differentiated in microfluidic chips show upregulation of neuroectodermal markers (ZIC1, PAX6, SOX1), highlighting their propensity for nervous system development. Cells obtained on conventional well plates and microfluidic chips are compared for reprogramming and neural induction by bulk RNA sequencing. Pathway enrichment analysis of NSCs from chip showed neural stem cell development enrichment and boosted commitment to neural stem cell lineage in initial phases of neural induction, attributed to a confined environment in a microfluidic chip. This method provides a cost-effective pipeline to reprogram and differentiate iPSCs for therapeutics compliant with current good manufacturing practices.

This study highlights the development of a microfluidic platform to reprogram somatic cells from donors into induced pluripotent stem cells and further differentiate them into neural stem cells. This confined microfluidic platform boosts neural stem cell generation commitment at an early stage, as denoted by the pathway enrichment analysis. image

Detection of bio-molecules through quantum tunneling currents could lead to the next-generation DNA sequencing methods. In order to analyze the stability of these sensitive devices, it is necessary to characterize their conductance switching statistics. This characterization can be realized by denoising the tunneling current signal and clustering the outcomes. The first step can be done with the CUSUM algorithm, which detects abrupt changes and has been used in similar devices. We found heavy-tailed non-Gaussian noise in the measurement setup of the experimental devices. This paper suggests an approximation in the likelihood ratio step of the CUSUM algorithm that is more robust than the simple Gaussian noise assumption and, at the same time, is computationally more efficient than computing the fitted true likelihoods.