Analytische Chemie
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- 1.5 Proteinanalytik (5) (entfernen)
BioPIC - Integration of Biosensors based on Photonic Integrated Circuits by Local-Backside Etching
(2020)
Silicon photonic sensors are promising candidates for lab-on-a-chip solutions with versatile applications and scalable production prospects using complementary metal-oxide semiconductor (CMOS) fabrication methods. However, the widespread use has been hindered because the sensing area adjoins optical and electrical components making packaging and sensor handling challenging. In this work, a local back-side release of the photonic sensor is employed, enabling a separation of the sensing area from the rest of the chip. This approach allows preserving the compatibility of photonic integrated circuits in the front-end of line and metal interconnects in the back-end of line.
CMOS-Compatible Silicon Photonic Sensor for Refractive Index Sensing Using Local Back-Side Release
(2020)
Silicon photonic sensors are promising candidates for lab-on-a-chip solutions with versatile applications and scalable production prospects using complementary metal-oxide semiconductor (CMOS) fabrication methods. However, the widespread use has been hindered because the sensing area adjoins optical and electrical components making packaging and sensor handling challenging. In this work, a local back-side release of the photonic sensor is employed, enabling a separation of the sensing area from the rest of the chip. This approach allows preserving the compatibility of photonic integrated circuits in the front-end of line and metal interconnects in the back-end of line. The sensor is based on a micro-ring resonator and is fabricated on wafer-level using a CMOS technology. We revealed a ring resonator sensitivity for homogeneous sensing of 106 nm/RIU.
The mechanism of this system is based on kinetic competition. This biosensor consists of a monolithic glass column with a vast excess of immobilized hapten, which traps the fluorescently labeled antibody as long as no explosive is present. If the explosive 2,4,6-trinitrotoluene (TNT) is introduced some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein. The fluorescence is detected by highly sensitive laser-induced fluorescence with a conventional CMOS camera. The system achieved limits of detection of approx.1 pM (1 ppt) of the fluorescent label and around 100 pM (20 ppt) of TNT. The total assay time is less than 8 minutes. A cross-reactivity test with 5000 pM solutions of pentaerythritol tetranitrate (PETN), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) showed no cross reactivity.
The illegal use of explosives by terrorists and other criminals is an increasing issue in public spaces, such as airports, railway stations, highways, sports venues, theaters, and other large buildings. Security in these environments can be achieved by different means, including the installation of scanners and other analytical devices to detect ultra-small traces of explosives in a very short time-frame to be able to take action as early as possible to prevent the detonation of such devices. Unfortunately, an ideal explosive detection system still does not exist, which means that a compromise is needed in practice. Most detection devices lack the extreme analytical sensitivity, which is nevertheless necessary due to the low vapor pressure of nearly all explosives. In addition, the rate of false positives needs to be virtually zero, which is also very difficult to achieve. Here we present an immunosensor system based on kinetic competition, which is known to be very fast and may even overcome affinity limitation, which impairs the performance of many traditional competitive assays. This immunosensor consists of a monolithic glass column with a vast excess of immobilized hapten, which traps the fluorescently labeled antibody as long as no explosive is present. In the case of the explosive 2,4,6-trinitrotoluene (TNT), some binding sites of the antibody will be blocked, which leads to an immediate breakthrough of the labeled protein, detectable by highly sensitive laser-induced fluorescence with the help of a Peltier-cooled complementary metal-oxide-semiconductor (CMOS) camera. Liquid handling is performed with high-precision syringe pumps and chip-based mixing-devices and flow-cells. The system achieved limits of detection of 1 pM (1 ppt) of the fluorescent label and around 100 pM (20 ppt) of TNT. The total assay time is less than 8 min. A cross-reactivity test with 5000 pM solutions showed no signal by pentaerythritol tetranitrate (PETN), 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). This immunosensor belongs to the most sensitive and fastest detectors for TNT with no significant cross-reactivity by non-related compounds. The consumption of the labeled antibody is surprisingly low: 1 mg of the reagent would be sufficient for more than one year of continuous biosensor operation.
The primary screening of hybridoma cells is a time-critical and laborious step during the development of monoclonal antibodies. Often, critical errors occur in this phase, which supports the notion that the generation of monoclonal antibodies with hybridoma technology is difficult to control and hence, a risky venture. We think that it is crucial to improve the screening process to eliminate most of the critical deficits of the conventional approach. With this new microarray-based procedure, several advances could be achieved: Selectivity for excellent binders, high-throughput, reproducible signals, avoidance of misleading avidity (multivalency) effects, and performance of simultaneous competition experiments. The latter can also be used to select clones of desired cross-reactivity properties. In this paper, a model system with two excellent clones against carbamazepine, two weak clones, and blank supernatant containing fetal bovine serum was designed to examine the effectiveness of the new system. The excellent clones could be detected largely independent of the immunoglobulin G (IgG) concentration, which is usually unknown during the clone screening since the determination and subsequent adjustment of the antibody concentration are not feasible in most cases. Furthermore, in this approach, the enrichment, isolation, and purification of IgG for characterization is not necessary. Raw cell culture supernatant can be used directly, even when fetal calf serum (FCS) or other complex media is used. In addition, an improved method for the oriented antibody-immobilization on epoxy-silanized slides is presented. Based on the results of this model system with simulated hybridoma supernatants, we conclude that this approach should be preferable to most other protocols leading to many false positives, causing expensive and lengthy elimination steps to weed out the poor clones.