wissenschaftliche Publikationen


Lock-In Pixel CMOS Image Sensor for Time-Resolved Fluorescence Readout of Lateral-Flow Assays

Alexander Hofmann, Benjamin Saft, Peggy Reich, Martin Grabmann, Georg Gläser, Max Trübenbach, Alexander Rolapp, Marco Reinhard, Friedrich Scholz, Eric Schäfer


We present a CMOS image sensor (CIS) based time-resolved fluorescence (TRF) measurement system for filter-less, highly sensitive readout of lateral-flow assay (LFA) test strips. The CIS contains a 256 × 128 lock-in pixel (LIP) sensor array. Each pixel has a size of 10μm × 10μm and includes a photodiode acting as signal transducer. The LIP CIS was designed in a standard 0.18-μm CMOS technology specifically for TRF applications. The LIP architecture blocks interfering light when fluorophores are excited and accumulates the emitted fluorescence light to be measured over multiple cycles after excitation. This allows to detect even small amounts of fluorescence light over a wide analyte concentration range. The LIP CIS based TRF reader was characterized in terms of reproducible and uniform signal intensities with use of appropriate Europium(III) [Eu 3+] chelate particles as fluorescence standards. We measured different concentrations of Eu-based nanoparticles (NP) on test strips with the TRF reader. The sensor system shows 5.1 orders of magnitude of detection dynamic range (DDR) with a limit of detection (LoD) of 0.1 ng/cm. In addition, using human C-reactive protein (hCRP) as a model analyte, we compared the developed TRF reader with a commercial colorimetric LFA reader. For the quantification of CRP, the LIP CIS based TRF reader demonstrates a DDR of 3.6 orders of magnitude with an excellent LoD of 0.05 ng/mL, which is 14 times better than the LoD of the commercial LFA reader.


Filterless TRF Reader with CMOS Sensor ASIC for Lateral Flow Immunoassays

Alexander Hofmann, Peggy Reich, Martin Grabmann, Georg Gläser, Alexander Rolapp, Marco Reinhard, Eric Schäfer, Max Trübenbach, Friedrich Scholz


In this paper, we present a filterless time-resolved fluorescence (TRF) measurement system with an optoelectronic CMOS sensor for highly sensitive readout of lateral flow assay (LFA) test strips. An application specific integrated circuit (ASIC) was designed in a standard 0.35-µm CMOS technology and includes a 5 × 5 CMOS sensor array. Each sensor pixel contains a 100 µm × 100 µm photodiode acting as signal transducer. For the precise control of the measurement sequence and its timing regime, a microcode engine (MCE) has been realized in a field-programmable gate array (FPGA). An active LED discharge concept was implemented to prevent long afterglow of the light source after it is turned off. To characterize the TRF reader with respect to uniform and reproducible signal intensities, suitable europium (III) [Eu 3+] chelate particles were used as fluorescence standards. Different concentrations of europium based nanoparticles (NP) on test strips were quantified with the TRF reader. Furthermore, we compared the developed TRF reader to a commercial colorimetric LFA reader system using human C-reactive protein (hCRP) as model analyte. The CMOS sensor ASIC based filterless TRF reader showed a detection dynamic range (DDR) of 4 orders of magnitude with a LoD of 0.2 ng/ml for the quantification of CRP, which was 3.5 times better than the LoD of the commercial LFA reader.

Light Absorption Measurement With a CMOS Biochip for Quantitative Immunoassay Based Point-of-Care Applications

Alexander Hofmann, Michael Meister, Alexander Rolapp, Peggy Reich, Friedrich Scholz, Eric Schafer


We present a CMOS biochip-based photometer for quantitative immunoassay diagnostics. The photometer quantifies the concentration of antigens based on light absorption, which allows for a low-cost implementation without expensive optical components. We propose a light controller to lower the start-up and settling time of the light source to 30 seconds, to facilitate fast measurement starts, and to decrease the overall measurement times. The application-specific integrated circuit (ASIC) contains a 6 x 7-sensor array with 100 μm x 100 μm photodiodes that serve as signal transducers. The ASIC was developed in a normal 0.35- μm CMOS technology, avoiding the need for expensive post-CMOS processes. We present our strategy for the assembly of the ASIC and the immobilization of antibodies. For its first time, we demonstrate the quantification of prostate specific antigen (PSA) with an optoelectronic CMOS biochip using this approach. A PSA immunoassay is performed on the top surface of the CMOS sensor array, enzyme kinetics and PSA concentration are measured within 6 minutes with a limit of detection (LoD) of 0.5 ng/ml, which meets clinical testing requirements. We achieve an overall coefficient of variation (CV) of 7%, which is good compared to other point-of-care (PoC) systems.


Carboxyl functionalized gold nanorods for sensitive visual detection of biomolecules

Friedrich Scholz, Lukas Rüttinger, Theresa Heckmann, Lisa Freund, Anne-Marie Gad, Tobias Fischer, Andreas Gütter, Hans Hermann Söffing


Antibody-modified gold nanomaterials are central to many novel biosensing technologies for example the lateral flow assays technology. The combination of the specificity, provided by antibody-antigen interactions, and the unique optical properties of nanomaterials provide excellent properties for biosensors. Here, we present the use of gold nanorods (GNR) with the localized Surface Plasmon Resonance (LSPR) peak in the visible range for biomarker detection. The colour of the GNR can be tuned by the reaction conditions to provide multi-coloured gold nanorod conjugates. These antibody functionalized GNR have the potential to provide significant improvements in multiplexed analysis and sensitivity compared to conventional gold nanoparticle based lateral flow assays. However, a major challenge is the synthesis of stable conjugates that resist aggregation in samples with high ionic strength, (e.g. salt solutions) and allow highly sensitive detection of proteins. A detailed investigation of different reagents for the functionalization of gold nanorod materials are reported. An antibody modified GNR based lateral flow assay is validated for the determination of C-reactive Protein (CRP).

Melioidosis DS rapid test: A standardized serological dipstick assay with increased sensitivity and reliability due to multiplex detection

Gabriel E Wagner, Esther Föderl-Höbenreich, Karoline Assig, Michaela Lipp, Andreas Berner, Christian Kohler, Sabine Lichtenegger, Julia Stiehler, Wisansanee Karoonboonyanan, Nida Thanapattarapairoj, Chidchanok Promkong, Sirikamon Koosakulnirand, Panjaporn Chaichana, Ralf Ehricht, Anne-Marie Gad, Hans H Söffing, Susanna J Dunachie, Narisara Chantratita, Ivo Steinmetz


Background: Melioidosis, caused by Burkholderia pseudomallei, is a severe infectious disease with high mortality rates, but is under-recognized worldwide. In endemic areas, there is a great need for simple, low-cost and rapid diagnostic tools. In a previous study we showed, that a protein multiplex array with 20 B. pseudomallei-specific antigens detects antibodies in melioidosis patients with high sensitivity and specificity. In a subsequent study the high potential of anti-B. pseudomallei antibody detection was confirmed using a rapid Hcp1 single protein-based assay. Our protein array also showed that the antibody profile varies between patients, possibly due to a combination of host factors but also antigen variations in the infecting B. pseudomallei strains. The aim of this study was to develop a rapid test, combining Hcp1 and the best performing antigens BPSL2096, BPSL2697 and BPSS0477 from our previous study, to take advantage of simultaneous antibody detection.
Methods and principal findings: The 4-plex dipstick was validated with sera from 75 patients on admission plus control groups, achieving 92% sensitivity and 97-100% specificity. We then re-evaluated melioidosis sera with the 4-plex assay that were previously misclassified by the monoplex Hcp1 rapid test. 12 out of 55 (21.8%) false-negative samples were positive in our new dipstick assay. Among those, 4 sera (7.3%) were Hcp1 positive, whereas 8 (14.5%) sera remained Hcp1 negative but gave a positive reaction with our additional antigens.
Conclusions: Our dipstick rapid test represents an inexpensive, standardized and simple diagnostic tool with an improved serodiagnostic performance due to multiplex detection. Each additional band on the test strip makes a false-positive result more unlikely, contributing to its reliability. Future prospective studies will seek to validate the gain in sensitivity and specificity of our multiplex rapid test approach in different melioidosis patient cohorts.

Light-Controlled Photometer with Optoelectronic CMOS Biochip for Quantitative PSA Detection

Alexander Hofmann, Michael Meister, Alexander Rolapp, Peggy Reich, Friedrich Scholz, Eric Schäfer


We present an integrated optoelectronic CMOS biochip based photometer for quantitative immunoassay diagnostics. The application specific integrated circuit (ASIC) includes a 6 × 7 CMOS sensor array with 100 μm × 100 μm photodiodes acting as signal transducers. The ASIC was designed in a standard 0.35-μm CMOS technology without expensive post-CMOS processes. The principle of the photometer is based on light absorption measurement. The implemented light control reduces start-up and settling time of the light source to 30 seconds, enables fast starts of measurements and reduces overall measurement times. With this approach, we demonstrate for the first time the quantification of prostate-specific antigen (PSA) using an optoelectronic CMOS biochip. A PSA immunoassay is performed on the top surface of the CMOS sensor array, enzyme kinetics and PSA concentrations are measured within 6minutes with a limit-of-detection (LoD) of 0.5 ng/ml. The LoD meets the requirement for clinical testing, since the cutoff level of PSA is 4 ng/ml. Additionally, we achieve an overall run-to-run coefficient of variations (CV) of 7% which is good compared to other point-of-care (PoC) systems.


An Integrated CMOS Photodiode Array for Highly Sensitive Photometric Diagnostics

Alexander Hofmann; Michael Meister; Susette Germer; Friedrich Scholz


This work describes the development and test of an integrated CMOS photodiode sensor array for bioanalytical applications. The integrated circuit (IC) was optimized for light absorption measurement and low-cost system integration. The CMOS-based measurement system was tested and compared to a commercial optical power meter. Opto-electrical measurement results prove a measurement range of three decades with a detection limit of the optical density OD = 0.001 in air at maximum sensitivity. Furthermore, we demonstrate the system usability for bioanalytical characterization performing a quantitative detection of gold nanoparticles (NP) deposited on the CMOS photodiodes. Therefore, measurements show a minimum detectable mean value of OD = 0.009, which corresponds to a small particle number of 38900 on top of the photodiode sensor surface. This CMOS based photometric measurement approach states the possibility to be used in low-cost, quantitative photometric diagnostic systems for medical applications e.g. for early cancer recognition.