Drug screening is widespread in contexts such as the criminal justice system, employment, and substance abuse treatment centers. Traditionally, drug testing methods have targeted urine matrices and extensive research is available regarding urine drug screening. Due to the nature of sample collection, urine specimen may be tampered or adulterated in efforts to invalidate or pass a drug test. Examples of this include substituting a sample with synthetic urine, diluting a sample with water, or adding a substance to the sample. The addition of a substance is referred to as adulteration and is done in an effort to mask drugs in the sample or to invalidate the test results. For various reasons, including the effects of adulteration, time, and costs associated with urine drug tests, oral fluid (OF) has become an increasingly important alternative matrix for screening drugs of abuse. It offers distinct advantages since tests can be administered noninvasively, quickly, and under observation, thus reducing the risk of tampering. Studies have also shown that drugs of abuse detected in OF may correlate better to user impairment at the time of collection, as compared to other matrices. In 2019, the Substance Abuse and Mental Health Service Administration (SAMHSA) published mandatory guidelines for oral fluid testing. Although these guidelines only directly impact federal spaces, they also serve as a blueprint for developing drug testing laws and policies in other organizations. Despite requirements and procedures controlling for specimen adulteration, it is recognized that manufacturers will continue to develop and market new products to avoid drug detection, just as with urine drug tests. The design of this experiment was to investigate the effects of the commercially available drug testing subversion products High Voltage Saliva Cleanse Mouthwash (High Voltage Detox, Las Vegas, Nevada, USA) and Stinger Detox Mouthwash (Stinger Detox, Phoenix, Arizona, USA) on immunoassay testing for tetrahydrocannabinol (THC) and methamphetamine (MET) in OF. The High Voltage Saliva Cleanse was designated adulterant “A”, and the Stinger Detox was designated adulterant “B”. Two separate immunoassay kits, Discover™ (American Screening Corporation, Inc., Shreveport, Louisiana, USA) and Orawell® (Jiangsu Well Biotech Co., Ltd, Changzhou, Jiangsu, China), were assessed to investigate the differences in performance of the current available test devices in addition to the effects of the subversion products. Using Discover™, samples were spiked according to 0.5 times (x), 1x, and 2x the cutoff concentrations of 50 ng/mL THC and 50 ng/mL MET without adulterant, with Adulterant A, and with Adulterant B. Testing with Orawell® devices was initially conducted at 1x and 2x the cutoff concentrations of 40 ng/mL THC and 50 ng/mL of MET. Based on the lack of response, testing at 0.5x was not conducted. Additional testing was conducted at 1.5x and 3x the cutoff concentrations without adulterant, with Adulterant A, and with Adulterant B. It was concluded that the adulterants affected the test results in the Orawell® device, by producing false positives for drugs of abuse not present in the sample for 17 (56.7%) of the 30 tests containing adulterants. Additionally, it was concluded that both immunoassay tests assessed were lacking in analytical sensitivity and reproducibility.

The manufacturing and the development of biotherapeutic drugs involves the expression of biotherapeutic proteins in a host cell expression system followed by a purification process. Bioanalytical methods to measure impurities such as host cell proteins (HCPs) are needed to obtain a robust process and a safe drug according to regulatory requirements. The aim of this project was to develop three specific HCP assays for detection and quantification of specific HCPsusing the Gyrolab® platform. The HCPs (Annexin A5, Clusterin and Nidogen-1) chosen for this project are generated from Chinese hamster ovary (CHO) cells. Each assay was evaluated on four different Gyrolab® BioaffyTM CDs with a comparison of column profiles, accuracy, precision and sensitivity. For each assay the best suited CD type was suggested together with possible upper limit of quantification (ULOQ) and lower limit of quantification (LLOQ) within the estimated detection range. The results indicate that the CHO Annexin A5 assay has a detection range extending from 1500 ng/ml to 2.1 ng/ml, with possible ULOQ at 1000 ng/ml and LLOQ at 3.2ng/ml using the BioaffyTM 4000 HC CD. The CHO Clusterin assay has a detection range extending from 1500 ng/ml to 0.4 ng/ml, with possible ULOQ at 1000 ng/ml and LLOQ at 0.7 ng/ml using the BioaffyTM 4000 HC CD. The CHO Nidogen-1 assay has a detection range extending from 1500 ng/ml to 0.1 ng/ml, with ULOQ at 1000 ng/ml and LLOQ at 0.2 ng/ml using the BioaffyTM1000 CD.

3D printing has gained substantial interest as an adaptable and low-cost technology for rapid prototyping and production of research tools owing to its fast design-to-object workflow (Fig. 1), ease of operation, and ability to fabricate relatively complex and intricate structures directly from computer-aided design (CAD) representations. Due to the advantages 3D printing offers over other more time-consuming and labor-intensive fabrication methods like photolithography, 3D printing has been especially helpful in the development and production of flow cells and other fluidic devices. 3D printing allows for complex channel geometries, and the complete structure, including ports for connecting commercially available tubing, may be prepared from a single CAD file. As a result of these conveniences, 3D-printed fluidic devices have recently emerged as effective candidates for research in sensing applications. In these studies, we demonstrate electrochemical immunoassays for the biomarker protein S100B, which has been related to conditions like skin cancer and brain injuries, based on 3D-printed flow-cells with modularly integrated electrodes. The fluidic devices in these studies are prepared from photocurable resin and feature channel dimensions of ~400 µm. The device design includes ports for interfacing the channel with commercial fittings and tubing for fluid delivery as well as an access point for the antibody-modified electrode. Sensing is accomplished through a sandwich-type electrochemical immunoassay strategy, leading to sensitive detection of S100B.

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Macrotroponin, formed by the complexation of endogenous cardiac troponin autoantibodies with circulating cardiac troponin, poses a challenge in high-sensitive assays generating high values without cardiac damage. Treatment of blood samples with polyethylene glycol (PEG) precipitates biomacromolecules, including macrotroponin, thereby freeing up smaller molecules, such as monomeric troponin in solution. While previous studies have predominantly examined the impact on recovery of PEG-precipitation on troponin I assays, this pilot study sought to explore its effects on routine samples using a troponin T assay and how the PEG-precipitation method affects the recovery of troponins and other proteins of varying sizes, including ferritin, albumin, TSH, cystatin C, prolactin, IgA, IgM, and IgG. PEG-precipitation was employed as the separation method and conducted on 20 different troponin T samples, with the yield of troponin and other proteins examined, and also on a series of dilutions of one and the same sample with initially high troponin concentration. To estimate the robustness of the PEG precipitation method, all samples were measured as duplicates.  For both the 20 individual samples and the diluted sample, the recovery of troponin decreased significantly with increasing troponin concentrations. This has not been previously documented in the literature. The finding implies that when employing PEG-precipitation to identify or rule out antibody-induced interference in troponin assays, it is imperative to account for the total concentration to evaluate the exchange rate. Furthermore, the PEG-precipitation method seems quite robust according to the low coefficient of variation for duplicate measurement.

This research presents a novel biosensor which utilizes the refractive index sensitivity of a fiber optic long-period grating. The long period grating couples light from the forward propagating guided core mode of a single-mode fiber into discrete circularly symmetric cladding modes. Due to imperfections in the cladding surface, loss bands are seen in the transmission spectrum corresponding to the coupled wavelengths. Based on the phase-matching condition between the coupling and coupled modes, the loss bands shift with changes in the refractive index of the surrounding medium. The grating surface is chemically treated to covalently bond antibody to the cladding of the sensor. Treatment with the proper antigen increases the effective index seen by the cladding modes and affects the placement of the loss bands. This sensor demonstrates specific antigen binding capacity with loss band shifts of 10 nm or more. The device offers several advantages over the widely used Enzyme-Linked Immuno-Sorbent Assays. Diagnostic applications can be expanded beyond the tests presented here. / Master of Science

Förster Resonance Energy Transfer (FRET) plays an important role for biochemical applications such as DNA sequencing, intracellular protein-protein interactions, molecular binding studies, in vitro diagnostics and many others. For qualitative and quantitative analysis, FRET systems are usually assembled through molecular recognition of biomolecules conjugated with donor and acceptor luminophores. Lanthanide (Ln) complexes, as well as semiconductor quantum dot nanocrystals (QD), possess unique photophysical properties that make them especially suitable for applied FRET. In this work the possibility of using QD as very efficient FRET acceptors in combination with Ln complexes as donors in biochemical systems is demonstrated. The necessary theoretical and practical background of FRET, Ln complexes, QD and the applied biochemical models is outlined. In addition, scientific as well as commercial applications are presented. FRET can be used to measure structural changes or dynamics at distances ranging from approximately 1 to 10 nm. The very strong and well characterized binding process between streptavidin (Strep) and biotin (Biot) is used as a biomolecular model system. A FRET system is established by Strep conjugation with the Ln complexes and QD biotinylation. Three Ln complexes (one with Tb3+ and two with Eu3+ as central ion) are used as FRET donors. Besides the QD two further acceptors, the luminescent crosslinked protein allophycocyanin (APC) and a commercial fluorescence dye (DY633), are investigated for direct comparison. FRET is demonstrated for all donor-acceptor pairs by acceptor emission sensitization and a more than 1000-fold increase of the luminescence decay time in the case of QD reaching the hundred microsecond regime. Detailed photophysical characterization of donors and acceptors permits analysis of the bioconjugates and calculation of the FRET parameters. Extremely large Förster radii of more than 100 Å are achieved for QD as acceptors, considerably larger than for APC and DY633 (ca. 80 and 60 Å). Special attention is paid to interactions with different additives in aqueous solutions, namely borate buffer, bovine serum albumin (BSA), sodium azide and potassium fluoride (KF). A more than 10-fold limit of detection (LOD) decrease compared to the extensively characterized and frequently used donor-acceptor pair of Europium tris(bipyridine) (Eu-TBP) and APC is demonstrated for the FRET system, consisting of the Tb complex and QD. A sub-picomolar LOD for QD is achieved with this system in azide free borate buffer (pH 8.3) containing 2 % BSA and 0.5 M KF. In order to transfer the Strep-Biot model system to a real-life in vitro diagnostic application, two kinds of imunnoassays are investigated using human chorionic gonadotropin (HCG) as analyte. HCG itself, as well as two monoclonal anti-HCG mouse-IgG (immunoglobulin G) antibodies are labeled with the Tb complex and QD, respectively. Although no sufficient evidence for FRET can be found for a sandwich assay, FRET becomes obvious in a direct HCG-IgG assay showing the feasibility of using the Ln-QD donor-acceptor pair as highly sensitive analytical tool for in vitro diagnostics. / Förster Resonanzenergietransfer (FRET) spielt eine wichtige Rolle in biochemischen Anwendungen, wie z.B. DNA-Sequenzierung, intrazellulären Protein-Protein-Wechselwirkungen, molekularen Bindungsstudien, in-vitro-Diagnostik und vielen anderen. Zur quantitativen und qualitativen Analyse werden FRET Systeme normalerweise durch molekulare Erkennung von Biomolekülen, die mit Donator- und Acceptorluminophoren markiert sind, ermöglicht. Durch die besonderen photophysikalischen Eigenschaften sowohl von Lanthanidkomplexen (Ln-Komplexen), als auch Halbleiternanokristallen (sog. Quantenpunkten oder Quantumdots - QD), sind diese besonders für FRET Anwendungen geeignet. In der vorliegenden Arbeit wird effizienter FRET zwischen Ln-Komplexen und QD in biochemischen Systemen demonstriert. Die notwendigen theoretischen und praktischen Grundlagen über FRET, Ln-Komplexe, QD und die verwendeten biochemischen Modelle werden dargestellt, und wissenschaftliche als auch kommerzielle Anwendungen werden präsentiert. FRET kann zur Messung von strukturellen Veränderungen und Dynamiken im Bereich von ca. 1 bis 10 nm verwendet werden. Der sehr starke und gut charakterisierte Bindungsprozess zwischen Streptavidin (Strep) und Biotin (Biot) wird als biomolekulares Modellsystem eingesetzt. Ein FRET System wird durch Streptavidinkonjugation mit Ln-Komplexen und QD-Biotinylierung etabliert. Drei Ln-Komplexe (einer mit Tb3+ und zwei mit Eu3+ als Zentralion) werden als Donatoren verwendet, und neben QD werden zwei weitere Acceptoren, das lumineszierende, quervernetzte Protein Allophycocyanin (APC) und ein kommerzieller Fluoreszenzfarbstoff (DY633), untersucht. FRET kann für alle Donator-Acceptor Paare nachgewiesen werden, zum einen durch sensibilisierte Acceptorlumineszenz und zum anderen durch eine über 1000-fach erhöhte Lumineszenzabklingzeit der QD mit über 100 Mikrosekunden. Mittels detailierter photophysikalischer Charakterisierung der Donatoren und Acceptoren können die Biokonjugate analysiert und die FRET Parameter berechnet werden. Für die QD FRET Systeme ergeben sich extrem große Försterradien von über 100 Å, die wesentlich größer sind als für APC und DY633 (ca. 80 bzw. 60 Å). Besondere Aufmerksamkeit gilt der Wechselwirkung mit den Zusatzreagenzien Boratpuffer, Bovines Serumalbumin (BSA), Natriumazid und Kaliumfluorid (KF) in den wässrigen Lösungen. Im Vergleich zum ausgiebig charakterisierten und vielfach verwendeten Donator-Acceptor Paar aus Europium-tris(Bipyridin) (Eu-TBP) und APC wird eine mehr als 10-fache Senkung der Nachweisgrenze für das FRET-System, bestehend aus Tb-Komplex und QD, erreicht. In azidfreiem Boratpuffer (pH 8,3) mit 2 % BSA und 0,5 M KF wird eine subpicomolare QD-Nachweisgrenze für dieses System aufgezeigt. Um den Transfer des Strep-Biot Modellsystems in eine echte in-vitro-diagnostische Anwendung zu demonstrieren, werden zwei Immuntests zum HCG-(Humanes Choriongonadotropin)-Nachweis untersucht. Sowohl HCG als auch monoklonale anti-HCG Maus-IgG-(Immunoglobulin G)-Antikörper werden mit dem Tb-Komplex bzw. mit QD markiert. Obwohl kein ausreichender Nachweis für FRET in einem immunometrischen Assay (oder Sandwichassay) erbracht werden kann, wird FRET in einem direkten HCG-IgG Assay erzielt, wodurch die Realisierbarkeit von Ln-QD Donator-Acceptor Paaren zur hochsensitiven Anwendung in der in-vitro-Diagnostik gezeigt werden kann.