Two examples of our biophotonic research utilizing nanoparticles are presented, namely laser-based fluoroimmuno analysis and in-vivo optical oxygen monitoring. Results of the work include significantly enhanced sensitivity of a homogeneous fluorescence immunoassay and markedly improved spatial resolution of oxygen gradients in root nodules of a legume species.

Quantum dots (QDs) are common as luminescing markers for imaging in biological applications because their optical properties seem to be inert against their surrounding solvent. This, together with broad and strong absorption bands and intense, sharp tuneable luminescence bands, makes them interesting candidates for methods utilizing Förster Resonance Energy Transfer (FRET), e. g. for sensitive homogeneous fluoroimmunoassays (FIA). In this work we demonstrate energy transfer from Eu<SUP>3+</SUP>-trisbipyridin (Eu-TBP) donors to CdSe-ZnS-QD acceptors in solutions with and without serum. The QDs are commercially available CdSe-ZnS core-shell particles emitting at 655 nm (QD655). The FRET system was achieved by the binding of the streptavidin conjugated donors with the biotin conjugated acceptors. After excitation of Eu-TBP and as result of the energy transfer, the luminescence of the QD655 acceptors also showed lengthened decay times like the donors. The energy transfer efficiency, as calculated from the decay times of the bound and the unbound components, amounted to 37%. The Förster-radius, estimated from the absorption and emission bands, was ca. 77 Å. The effective binding ratio, which not only depends on the ratio of binding pairs but also on unspecific binding, was obtained from the donor emission dependent on the concentration. As serum promotes unspecific binding, the overall FRET efficiency of the assay was reduced. We conclude that QDs are good substitutes for acceptors in FRET if combined with slow decay donors like Europium. The investigation of the influence of the serum provides guidance towards improving binding properties of QD assays.

In den letzten Jahren gerieten phenolische Substanzen, wie z.B. Chlor-, Nitrophenol oder Alkylphenolethoxylate aufgrund ihrer Toxizität sowie ihres kanzerogenen und endokrinen Potentials in das Interesse der Öffentlichkeit. Diese Substanzen gelangen in großen Mengen, z.B. aus industriellen Prozessen (Papier-, Kunststoff-, oder Lederindustrie) oder als Abbauprodukte von Pflanzenschutzmitteln in die Umwelt.<br /> <br /> Ziel dieser Arbeit war es, einfache biochemische Bestimmungsmethoden für verschiedene phenolische Umweltschadstoffe auf Basis biochemischer Erkennungselemente zu entwickeln. Diese sollten als Screeningmethoden in der Vor-Ort-Analytik einsetzbar sein. Die Anwendung sollte kostengünstig und einfach durchzuführen sein, so dass die Messung kein hochwissenschaftliches Personal erfordert. Daher stand im Hintergrund der Arbeit die Integration der Analysenmethode in ein kompaktes Handgerät.<br /> <br /> Zu diesem Zweck wurde ein Biosensor entwickelt der zur direkten Messung und in Kombination mit einem Immunoassay einsetzbar ist:<br /> <br /> 1.) Elektrochemischer Biosensor<br /> Ein elektrochemischer Biosensor stellt die Verbindung zwischen einer Elektrode und der biologischen Komponente dar. Als Messprinzip wurde die Amperometrie gewählt. Hierbei wird die Präsenz des nachzuweisenden Stoffes durch die angelegte Spannung am Sensor visualisiert, da beim Vorhandensein ein Stromfluss gemessen wird.<br /> <br /> Um die Signalintensität zu erhöhen können Enzyme als Katalysatoren genutzt werden, die in der Lage sind die Rückreaktion der Elektrodenreaktion zu realisieren. In diesem Fall wurde Glucose-Dehydrogenase (GDH) verwendet, die oxidierte phenolische Verbindungen reduzieren kann. Zusammen mit der Oxidation an der Sensoroberfläche bildet sich ein Verstärkungszyklus aus, der das ursprüngliche Signal vielfach erhöht.<br /> <br /> Wir waren in der Lage, GDH durch Einbetten in ein Polymerennetzwerk auf der Oberfläche einer gedruckten Platin-Dickschicht-Elektrode zu immobilisieren. Als Resultat erhielten wir einen sehr empfindlichen und äußerst stabilen Biosensor. Seine schnelle Ansprechzeit ermöglicht den Einsatz in automatisierten Fließsystemen zur Messung großer Probenzahlen. Der Einsatz in einem manuell betriebenen Handgerät konnte ebenfalls realisiert werden und brachte nur geringe Beeinträchtigungen in bezug auf die Empfindlichkeit der Messung. Die erfolgreiche Implementierung des Biosensors in das Handgerät wurde in Rahmen eines internationalen Workshops in Barcelona, anhand der Überprüfung der Reinigungsleistung von Klärwerken, gezeigt.<br /> <br /> 2.) Kombination mit Immunoassays<br /> Der Einsatzbereich der GDH-Biosensoren lässt sich durch die Kombination mit anderen Techniken erweitern, wobei der Sensor zur Visualisierung der Nachweisreaktion dient. In diesem Fall kann der Sensor zur Bestimmung der Enzymaktivität von ß?Galactosidase (ßGal) verwendet werden. Der Nachweis geringster Enzymmengen wurde realisiert. Die ßGal wird zur Markierung eines Analytanalogen in Immunoassays verwendet, um die Bindung von Antikörper und Analytmolekül sichtbar zu machen. Im Immunoassay bildet sich ein Gleichgewicht zwischen Antikörper, unmarkiertem Analyt und markiertem Analytanalog (Tracer) aus. Über die Bestimmung der Enzymaktivität kann man die Analytkonzentration in der Probe errechnen.<br /> <br /> Wir haben unseren GDH-Biosensor erfolgreich mit zwei Techniken kombiniert. Zum Einen mit einem Assay zur Bestimmung von Nitrophenol, der in einem automatisiertem Fließsystem realisiert wurde. Hier wird die Mischung aus Antikörpern, Analyt und Tracer über eine Säule gegeben und gespült. Die gebundenen Bestandteile werden durch den GDH-Biosensor quantifiziert.<br /> <br /> Zum Anderen wurde ein Kapillarimmunoassay entwickelt, der in das Handgerät integriert werden kann. Dabei wird der Antikörper direkt an der Kapillare fixiert. Die Probe wird mit Tracer vermischt und in die Kapillare gegeben. Dort bildet sich das Gleichgewicht aus und weitere Probenbestandteile werden im Spülschritt eliminiert. Die Analytkonzentration wird durch die Bestimmung des gebunden Tracers (Aktivität der ßGal) mit Hilfe des GDH-Biosensors realisiert. / The development of fast and reliable biochemical tools for on-site screening in environmental analysis was the main target of the present work. Due to various hazardous effects such as endocrine disruption and toxicity phenolic compounds are key analytes in environmental analysis and thus were chosen as model analytes.<br /> <br /> Three different methods were developed:<br /> <br /> For the enzymatic detection of phenols in environmental samples an enzyme-based biosensor was developed. In contrast to reported work using tyrosinase or peroxidases, we developed a biosensor based on glucose dehydrogenase as biorecognition element. This biosensor was devoted for an application in a laboratory flow system as well as in a portable device for on-site measurements.<br /> <br /> This enzymatic detection is applicable only for a limited number of phenols due to substrate specificity of the enzyme. For other relevant compounds based on a phenolic structure (i.e. nitrophenol, alkylphenols and alkylphenol ethoxylates) immunological methods had to be developed. The electrochemical GDH-biosensor was used as the label detector in these immunoassays.<br /> <br /> Two heterogeneous immunoassays were developed where ßGal was used as the label. An electrochemical method for the determination of the marker enzyme activity was processed. The separation step was realized with protein A/G columns (laboratory flow system) or by direct immobilization of the antibodies in small disposable capillaries (on-site analysis). <br /> <br /> All methods were targeted on the contemporary analysis of small numbers of samples.

The present study deals with the comparison and establishment of methods for the detection of antibodies against recombinant human growth hormone (rhGH). Therefore, different methods for the detection of hGH-Abs were evaluated and compared in order to establish a test system that can be used for the detection of neutralizing antibodies against hGH, which could be developed under rhGH treatment. This manuscript describes in detail the validation of a newly developed biological assay, the neutralizing hGH-antibody assay (NAb assay). Therefore, a cell line transfected with the growth hormone receptor, that proliferates in the presence of hGH, was used. This proliferation was quantified by an increase of the optical density (OD/ absorbance) after addition of a colorimetric reagent, whereas the presence of hGH-antibodies leads to an inhibition of cell proliferation. To validate the test system for the detection of hGH-antibodies, we tested serum samples of 4 patients suffering from neurosecretory dysfunction (NSD) and samples taken from 6 patients with growth hormone deficiency (GHD) which were treated with rhGH and were highly suspected for a-hGH antibodies. These samples were tested in two different immunological assays, capable to screen sera for anti-hGH immunreactivity in the case of hGH-insensitivity during GH treatment. Using the NAb assay the neutralizing activity of specific hGH-antibodies was proved in serum samples of NSD and GHD type 1A patients. In case of neutralizing hGH-antibody activity, a clinically based decision can be made whether rhGH therapy should be stopped or the rhGH dosis should be increased. By the use of our test system, we offer the measurement of anti-hGH-antibody activity to other laboratories in cases when secondary hGH-insensitivity is assumed or observed.

Van Kempen et al. described high natalizumab concentrations in their natalizumab-treated multiple sclerosis (MS) patients at time of re-dosing. Based on the literature research the authors consider a natalizumab concentration above 2 μg/mL to be sufficient for an adequate alpha-4 integrin receptor saturation of above 70%. Recently, we have demonstrated similar results using our new cell-based immunoassay to evaluate free natalizumab concentration, cell-bound natalizumab, and alpha-4 integrin receptor saturation as the key pharmacokinetic/pharmacodynamic parameters of natalizumab treatment in different in vivo settings. We investigated the effects of treatment interval extension or treatment cessation.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Ying Fang / The Porcine Disease Complex (PDC) results in major economic problems for swine producers. PDC outbreaks result in increased mortality, decreased feed efficiency, higher cull rates, prolonged days to market and increased treatment costs. This disease involves the interaction and participation of many multifactorial etiologies including both bacterial and viral organisms playing a role in disease initiation and progression. The most common viral pathogens associated with the PDC include porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus (PCV2) and swine influenza virus (swIV). The recent outbreak of porcine epidemic diarrhea virus (PEDV) in the US swine herd has made the PDC even more complicated. In aid of the prevention and control of the PDC, veterinarians and producers require fast and efficient diagnostic tests for controlling the disease. In this study, we have generated recombinant nucleocapsid antigens to these viruses for use in a Luminex™ technology-based fluorescent microsphere immunoassay (FMIA). Utilizing these recombinant nucleocapsid antigens, the FMIA was developed to simultaneously detect antibodies in serum from animals infected with PEDV, PRRSV, SwIV and PCV2. The FMIA was developed based on testing experimentally derived standard positive and negative control sera, and the diagnostic specificity and sensitivity were compared to that generated from the classical enzyme-linked immunosorbent assay (ELISA) or hemagglutination inhibition (HI) test. Based on an evaluation of 4147 serum samples with known serostatus, the multiplex FMIAs reached greater than 97.5% sensitivity and 92.3 % specificity. Results showed that multiplexing did not affect the diagnostic sensitivity or specificity of each individual assay. This work provides a platform for the development of multiplex assays for detecting various swine pathogens simultaneously and aids in preventing and controlling the PDC.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

This work introduces technology for rapid pathogen detection using handheld optical immunoassay and wire-guided droplet PCR systems. There have been a number of cases of foodborne or waterborne illness among humans that are caused by pathogens such as Escherichia coli O157:H7, Salmonella typhimurium, Influenza A H1N1, and the norovirus. The current practices to detect such pathogenic agents are: cell/viral culturing, immunoassays, or polymerase chain reactions (PCRs). These methods are essentially laboratory-based methods that are not at all real-time and thus unavailable for early-monitoring of such pathogens. They are also very difficult to be implemented in field, preventing early detection opportunities. This dissertation is divided into three papers that present methodologies towards the expeditious detections of infectious pathogens and the miniaturization of these detection systems towards field-deployable and point-of-care applications. Specifically, the work presented focuses on two methodologies: (1) light scatter detection using immunoagglutination assays with optimized Mie light scatter parameters in a real biological matrix consisting of plant tissue, and (2) wire-guided droplet manipulations for rapid and improved sample analysis, preparation, and PCR thermocycling. Both of these methods carry a collective objective towards providing high impact technologies for addressing the issues of food-related outbreaks and overall public safety. In the first paper, the direct and sensitive detection of foodborne pathogens from fresh produce samples was accomplished using a handheld lab-on-a-chip device, requiring little to no sample processing and enrichment steps for a near-real-time detection and truly field-deployable device. The detection of Escherichia coli K12 and O157:H7 in iceberg lettuce was achieved utilizing optimized Mie light scatter parameters with a latex particle immunoagglutination assay. The system exhibited good sensitivity, with a limit of detection of 10 CFU mL⁻¹ and an assay time of <6 min. Minimal pretreatment with no detrimental effects on assay sensitivity and reproducibility was accomplished with a simple and cost-effective KimWipes filter and disposable syringe. Mie simulations were used to determine the optimal parameters (particle size d, wavelength λ, and scatter angle θ) for the assay that maximize light scatter intensity of agglutinated latex microparticles and minimize light scatter intensity of the tissue fragments of iceberg lettuce, which were experimentally validated. This introduces a powerful method for detecting foodborne pathogens in fresh produce and other potential sample matrices. The integration of a multi-channel microfluidic chip allowed for differential detection of the agglutinated particles in the presence of the antigen, revealing a true field-deployable detection system with decreased assay time and improved robustness over comparable benchtop systems. In the second paper, we demonstrate a novel method of wire-guided droplet manipulations towards very quick RT-PCR. Because typical RT-PCR assays take about 1–2 h for thermocycling, there is a growing need to further speed up the thermocycling to less than 30 min. Additionally, the PCR assay system should be made portable as a point- of-care detection tool. Rapid movements of droplets (immersed in oil) over three different temperature zones make very quick PCR possible, as heating/cooling will be made by convective heat transfer, whose heat transfer coefficients are much higher than that of conduction, the latter of which is used in most conventional PCR systems. A 30-cycle PCR of a 160 bp gene sequence amplified from 2009 H1N1 influenza A (human origin) was successfully demonstrates in 6 min and 50 sec for a very large 10 μL droplet (with additional 4 min for reverse transcription). The proposed system has a potential to become a rapid, portable, point-of-care tool for detecting influenza A. In the third paper, a wire-guided CNC apparatus was used to perform droplet centrifugation, DNA extraction, and VQ-PCR thermocycling on a single superhydrophobic surface measuring 25 mm by 55 mm and a multi-chambered PCB heater. This methodology exhibited no limitations on the complexity and configuration of procedures that it can perform, making it versatile and far-reaching in its applications. The only modification required for adding or implementing changes for a new protocol is through simple pre-defined programming. The highly adaptive and flexible system was used to execute easily pre-programmed droplet movements and manipulations for the rapid detection of Escherichia coli from PCR detection. Serial dilutions were performed to simulate a diluted field sample with a high level of accuracy. Centrifugation of the diluted sample containing E. coli demonstrated a novel approach to sample pre-treatment. Furthermore, the extraction of DNA from the sample droplet containing E. coli was also performed on the same superhydrophobic surface as the previous 2 steps, requiring less than 10 min. Following extraction, the genetic material was amplified using wire-guided droplet PCR thermocycling, successfully completing 30 cycles of Peptidase D (a long 1500 bp sequence) in 10 min. The droplet centrifugation process was determined to greatly improve the positive band intensity over the non-centrifuged sample. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

Les analyses physico-chimiques des arômes du vin prennent aujourd’hui un essor considérable pour faciliter la prise de décision des professionnels de la vigne et du vin. Des analyses performantes, pour un certain nombre de molécules parmi les plus pertinentes, ont été développées (GC-MS, LC-MS). Cependant les techniques utilisées ne sont pas facilement transposables au sein des exploitations. L’objectif de cette thèse est donc de proposer un nouveau type de dosage peu onéreux et simple à mettre en œuvre. La technique envisagée est le dosage immunologique (tests ELISA) qui permet, pour quelques Euros, de doser directement sur le terrain les molécules pertinentes sans préparation préalable des échantillons. Les molécules visées (alkylméthoxypyrazines et précurseurs cystéinylés de thiols volatiles) sont présentes dans les vins de Cabernet Sauvignon et de Sauvignon blanc. / Analytical methods for the detection and quantification of wines aroma typically utilise HPLC-MS or GC-MS. The methods require some isolation and concentration step preceding the analysis. Enzyme-linked immunosorbent assays (ELISAs) are becoming either alternative complementary analytical tools to conventional methods because of their rapidity, sensitivity, selectivity, and low cost. In this Thesis, the applicability of ELISAs for detection and quantification of precursors of volatile thiols and alkylmethoxypyrazines, which have been isolated from wines, made from Cabernet Sauvignon or Sauvignon Blanc, are described.

Luminescent semiconductor quantum dots (QDs) are extensively researched for use in biological applications. They have unique optical and physical properties that make them excellent candidates to replace conventional organic dyes for cellular labeling, multiplexing, nucleic acid detection, and as generalized probes. The primary focus of this dissertation was to utilize quantum dots for improvement in immunoassays. Specifically, atherosclerosis biomarkers were detected simultaneously in an effort to demonstrate advances in early detection diagnostics. Quantum dot-antibody bioconjugates were prepared by encapsulation into mesoporous silica and functionalized with thiol and amine groups to enable bioconjugation. Functionalization of the mesoporous silica quantum dot composites facilitated biocompatibility for use with biological buffers in immunoassays. These bioconjugates were used in a sandwich immunoassay to detect atherosclerosis biomarkers IL-15 and MCP-1. Sandwich assays employ capture antibodies immobilized onto a well plate to bind as much of the antigen as possible. The capture antibodies increased binding by at least 4 times the amount of antigen bound to the surface of a direct detection assay. The sandwich immunoassay was able to detect 1 pg/mL of IL-15 and 50 pg/mL of MCP-1 biomarkers. Human serum albumin nanoparticles (HSAPs) were synthesized via a desolvation and crosslinking method. Human serum albumin is a versatile protein being used in a variety of applications. Quantum dots were loaded into HSAPs as potential detection probes for immunoassays. Efficient loading was not achieved, and the assay was unable to improve current detection limits. Controlled release studies were explored using HSAPs loaded with superparamagnetic iron oxide nanoparticles and a fluorescent drug analog. Exposure to a magnetic field resulted in degradation of the HSAPs. The fluorophore was released and measured to examine how cancer drugs might be controlled through a magnetic field. Gold nanorods and an anticancer drug, Sorafenib, were also encapsulated into HSAPs for treatment of renal cell carcinoma in vivo. Laser irradiation treatment combined with Sorafenib resulted in 100% tumor necrosis and total elimination of any viable tumor present. HSAPs have demonstrated remarkable potential as drug delivery nanocarriers.