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

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 10 µm × 10 µm and includes a photodiode acting as signal transducer. The LIP CIS was designed in a standard 0.18 µ m 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 [Formula: see text]. 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 [Formula: see text], which is 14 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.

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.

Impedimetric Aptamer-Based Biosensors: Principles and Techniques.

Aptamers are a specific class of ligands with high affinities comparable to antibodies, which are selected and synthesized in vitro. In combination with impedance spectroscopy as sensitive measurement method, we gain a class of biosensors with high potential for handheld devices and point-of-care tests. In this review, we report on recent advances in aptamer-based impedimetric biosensors. Besides giving a short summary of electrochemical measurement techniques, the most exciting innovative developments of detection strategies in the last decades are reviewed. Finally, important criteria for the comparison of aptamer-based biosensors are discussed.

Impedimetric Aptamer-Based Biosensors: Applications.

Impedimetric aptamer-based biosensors show high potential for handheld devices and point-of-care tests. In this review, we report on recent advances in aptamer-based impedimetric biosensors for applications in biotechnology. We detail on analytes relevant in medical and environmental biotechnology as well as food control, for which aptamer-based impedimetric biosensors were developed. The reviewed biosensors are examined for their performance, including sensitivity, selectivity, response time, and real sample validation. Additionally, the benefits and challenges of impedimetric aptasensors are summarized.

An aptamer-based biosensor for detection of doxorubicin by electrochemical impedance spectroscopy.

An aptamer-based biosensor was developed for the detection of doxorubicin using electrochemical impedance spectroscopy. Doxorubicin and its 14-dehydroxylated version daunorubicin are anthracyclines often used in cancer treatment. Due to their mutagenic and cardiotoxic effects, detection in groundwater is desirable. We developed a biosensor using the daunorubicin-binding aptamer as biological recognition element. The aptamer was successfully co-immobilized with mercaptohexanol on gold and a density of 1.3*1013 ± 2.4*1012 aptamer molecules per cm2 was achieved. The binding of doxorubicin to the immobilized aptamer was detected by electrochemical impedance spectroscopy. The principle is based on the inhibition of electron transfer between electrode and ferro-/ferricyanide in solution caused by the binding of doxorubicin to the immobilized aptamer. A linear relationship between the charge transfer resistance (R ct ) and the doxorubicin concentration was obtained over the range of 31 nM to 125 nM doxorubicin, with an apparent binding constant of 64 nM and a detection limit of 28 nM. With the advantages of high sensitivity, selectivity, and simple sensor construction, this method shows a high potential of impedimetric aptasensors in environmental monitoring. Graphical abstract Measurement chamber and immobilization principle for the detection of doxorubicin by electrochemical impedance spectroscopy.

Development of An Impedimetric Aptasensor for the Detection of Staphylococcus aureus.

In combination with electrochemical impedance spectroscopy, aptamer-based biosensors are a powerful tool for fast analytical devices. Herein, we present an impedimetric aptasensor for the detection of the human pathogen Staphylococcus aureus. The used aptamer targets protein A, a surface bound virulence factor of S. aureus. The thiol-modified protein A-binding aptamer was co-immobilized with 6-mercapto-1-hexanol onto gold electrodes by self-assembly. Optimization of the ratio of aptamer to 6-mercapto-1-hexanol resulted in an average density of 1.01 ± 0.44 × 1013 aptamer molecules per cm². As shown with quartz crystal microbalance experiments, the immobilized aptamer retained its functionality to bind recombinant protein A. Our impedimetric biosensor is based on the principle that binding of target molecules to the immobilized aptamer decreases the electron transfer between electrode and ferri-/ferrocyanide in solution, which is measured as an increase of impedance. Microscale thermophoresis measurements showed that addition of the redox probe ferri-/ferrocyanide has no influence on the binding of aptamer and its target. We demonstrated that upon incubation with various concentrations of S. aureus, the charge-transfer resistance increased proportionally. The developed biosensor showed a limit of detection of 10 CFU·mL-1 and results were available within 10 minutes. The biosensor is highly selective, distinguishing non-target bacteria such as Escherichia coli and Staphylococcus epidermidis. This work highlights the immense potential of impedimetric aptasensors for future biosensing applications.

Rapid and label-free detection of protein a by aptamer-tethered porous silicon nanostructures.

Protein A, which is secreted by and displayed on the cell membrane of Staphylococcus aureus is an important biomarker for S. aureus. Thus, its rapid and specific detection may facilitate the pathogen identification and initiation of proper treatment. Herein, we present a simple, label-free and rapid optical biosensor enabling specific detection of protein A. Protein A-binding aptamer serves as the capture probe and is immobilized onto a nanostructured porous silicon thin film, which serves as the optical transducer element. We demonstrate high sensitivity of the biosensor with a linear detection range between 8 and 23µM. The apparent dissociation constant was determined as 13.98µM and the LoD is 3.17µM. Harnessing the affinity between protein A and antibodies, a sandwich assay format was developed to amplify the optical signal associated with protein A capture by the aptamer. Using this approach, we increase the sensitivity of the biosensor, resulting in a three times lower LoD.