Single Photon Avalanche Diode Arrays for Time-Resolved Raman Spectroscopy.

The detection of peaks shifts in Raman spectroscopy enables a fingerprint reconstruction to discriminate among molecules with neither labelling nor sample preparation. Time-resolved Raman spectroscopy is an effective technique to reject the strong fluorescence background that profits from the time scale difference in the two responses: Raman photons are scattered almost instantaneously while fluorescence shows a nanoseconds time constant decay. The combination of short laser pulses with time-gated detectors enables the collection of only those photons synchronous with the pulse, thus rejecting fluorescent ones. This review addresses time-gating issues from the sensor standpoint and identifies single photon avalanche diode (SPAD) arrays as the most suitable single-photon detectors to be rapidly and precisely time-gated without bulky, complex, or expensive setups. At first, we discuss the requirements for ideal Raman SPAD arrays, particularly focusing on the design guidelines for optimized on-chip processing electronics. Then we present some existing SPAD-based architectures, featuring specific operation modes which can be usefully exploited for Raman spectroscopy. Finally, we highlight key aspects for future ultrafast Raman platforms and highly integrated sensors capable of undistorted identification of Raman peaks across many pixels.

Fluorescence-suppressed time-resolved Raman spectroscopy of pharmaceuticals using complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector.

In this work, we utilize a short-wavelength, 532-nm picosecond pulsed laser coupled with a time-gated complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector to acquire Raman spectra of several drugs of interest. With this approach, we are able to reveal previously unseen Raman features and suppress the fluorescence background of these drugs. Compared to traditional Raman setups, the present time-resolved technique has two major improvements. First, it is possible to overcome the strong fluorescence background that usually interferes with the much weaker Raman spectra. Second, using the high photon energy excitation light source, we are able to generate a stronger Raman signal compared to traditional instruments. In addition, observations in the time domain can be performed, thus enabling new capabilities in the field of Raman and fluorescence spectroscopy. With this system, we demonstrate for the first time the possibility of recording fluorescence-suppressed Raman spectra of solid, amorphous and crystalline, and non-photoluminescent and photoluminescent drugs such as caffeine, ranitidine hydrochloride, and indomethacin (amorphous and crystalline forms). The raw data acquired by utilizing only the picosecond pulsed laser and a CMOS SPAD detector could be used for identifying the compounds directly without any data processing. Moreover, to validate the accuracy of this time-resolved technique, we present density functional theory (DFT) calculations for a widely used gastric acid inhibitor, ranitidine hydrochloride. The obtained time-resolved Raman peaks were identified based on the calculations and existing literature. Raman spectra using non-time-resolved setups with continuous-wave 785- and 532-nm excitation lasers were used as reference data. Overall, this demonstration of time-resolved Raman and fluorescence measurements with a CMOS SPAD detector shows promise in diverse areas, including fundamental chemical research, the pharmaceutical setting, process analytical technology (PAT), and the life sciences.

Synthetic minority oversampling and iterative fluorescence-suppression integrated algorithm for Raman spectrum pesticide detection system.

Raman spectrum preprocessing method for automatic denoising and suppression of the fluorescent background. In this method, noise is reduced using wavelet transform, and a modified polynomial curve fitting method is implemented such that an algorithm can independently identify the optimal curve parameters for fluorescent background suppression. To address the problem of imbalanced datasets, the present study employed a synthetic minority oversampling technique to increase the volume of data in minority classes. This technique enables the prediction of pesticides that are otherwise difficult to detect, and the prediction accuracy is comparable to that of detection with large data volumes. The proposed convolutional neural network model was verified to accurately identify the type of single pesticides and composition of mixed pesticides. The prediction accuracy for mixed pesticides reached 99.1%.

An optimized green preparation method for the successful application of Raman spectroscopy in honey studies.

Raman spectroscopy represents an emerging technique for food authentication being a fast, reliable analytical method, requiring a minimum sample preparation step. Anyway, as in the case of any analytical techniques, there are some limitations which need to be properly assessed before applying this method in honey authentication. In this regard, the aim of this study consisted in the development of a simple working protocol, for honey sample preparation, which can simultaneously overcome the main limitations of Raman spectroscopy in honey studies, such as crystallization and fluorescence. Thus, in this work, a new green sample preparation method is proposed, discussed and its robustness is tested. It has been demonstrated that through honey dilution, in distilled water, reliable and reproductible spectra could be obtained, allowing the investigation of different types of honey. The main advantage of the method consists in the simultaneously overcoming of the most significant limitations of Raman spectroscopy employment in honey studies, such as crystallization and fluorescence, by a simple 1:1 w/v dilution of honey in distilled water.

Chemical Bleaching to Minimize Fluorescence Interference in Raman Spectroscopic Measurements for Sulfonated Polystyrene Solutions.

Auto-fluorescence is a significant challenge for Raman spectroscopic analyses. Since fluorescence is a much stronger phenomenon than Raman scattering, even trace fluorescent impurities can overwhelm the Raman signal. Strategies to minimize fluorescence interference in Raman measurements include either an instrumental-based approach or treatment of the sample itself to minimize fluorescence. Efforts focused on sample-based treatments to reduce fluorescence interferences have generally focused on sample purification and photobleaching methodologies. In this work, we present a sample treatment approach based upon chemical bleaching to remove fluorescence from Raman measurements of aqueous solutions of sulfonated polystyrene (SPS). Synthetic batches of SPS are characterized by a wide variation in fluorescence from minimum to a catastrophic level, which greatly limits the use of Raman spectroscopy. We systematically investigate the efficacy of various sample-based treatments of the SPS samples. An important acceptance criterion is that the procedure effectively and reliably removes fluorescence without damaging the SPS component. The chemical bleaching, which involves the addition of hydrogen peroxide and incubation at 60 ℃, is found to be highly effective. The parameters affecting the bleaching efficacy are studied, including temperature, hydrogen peroxide dosage, and bleaching time. Classification models are then developed based on the drastically diverse fluorescence background levels in Raman spectra of SPS to help optimize bleaching time for each specific sample. This work serves as an example of using chemical bleaching to remove fluorescence, which is inexpensive and readily available. It can facilitate a broader use of Raman spectroscopy as a quantitative qualitative control method in industrial settings.

Human alpha-fetal protein immunoassay using fluorescence suppression with fluorescent-bead/antibody conjugate and enzymatic reaction.

The aim of the study was to develop a simple and rapid immunoassay using fluorescent microbeads and enzyme-substrate reactions to measure alpha-fetal protein (AFP) concentrations. We demonstrated the functionality of the fluorescent immunosensor using antibody-conjugated fluorescent latex beads (AB-FLBs) and horseradish peroxidase (HRP) to catalyze a reaction, where the products would precipitate and suppress the fluorescence of AB-FLBs. First, the AB-FLBs were incubated with antigen, biotinylated antibodies (bABs), and streptavidin-HRP (SAv-HRP) to form a sandwich-type immunoreaction. The mixture was then filtered through a membrane to concentrate the beads on a small area. After washing to remove unbound bABs and SAv-HRP, a chromogenic HRP substrate and H2O2 were added to form precipitates on the FLB surface. The suppression of the fluorescence was measured with a fluorescent image analyzer system. Under optimized conditions, AFP could be measured at concentrations as low as 1 pg mL(-1) with a dynamic range up to 100 ng mL(-1).