Small Reagent Research Articles

Reagents for Mass Cytometry.

Mass cytometry (cytometry by time-of-flight detection [CyTOF]) is a bioanalytical technique that enables the identification and quantification of diverse features of cellular systems with single-cell resolution. In suspension mass cytometry, cells are stained with stable heavy-atom isotope-tagged reagents, and then the cells are nebulized into an inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS) instrument. In imaging mass cytometry, a pulsed laser is used to ablate ca. 1 μm2 spots of a tissue section. The plume is then transferred to the CyTOF, generating an image of biomarker expression. Similar measurements are possible with multiplexed ion bean imaging (MIBI). The unit mass resolution of the ICP-TOF-MS detector allows for multiparametric analysis of (in principle) up to 130 different parameters. Currently available reagents, however, allow simultaneous measurement of up to 50 biomarkers. As new reagents are developed, the scope of information that can be obtained by mass cytometry continues to increase, particularly due to the development of new small molecule reagents which enable monitoring of active biochemistry at the cellular level. This review summarizes the history and current state of mass cytometry reagent development and elaborates on areas where there is a need for new reagents. Additionally, this review provides guidelines on how new reagents should be tested and how the data should be presented to make them most meaningful to the mass cytometry user community.

Monodisperse Micro-Droplet Generation in Microfluidic Channel with Asymmetric Cross-Sectional Shape.

Micro-droplets are widely used in the fields of chemical and biological research, such as drug delivery, material synthesis, point-of-care diagnostics, and digital PCR. Droplet-based microfluidics has many advantages, such as small reagent consumption, fast reaction time, and independent control of each droplet. Therefore, various micro-droplet generation methods have been proposed, including T-junction breakup, capillary flow-focusing, planar flow-focusing, step emulsification, and high aspect (height-to-width) ratio confinement. In this study, we propose a microfluidic device for generating monodisperse micro-droplets, the microfluidic channel of which has an asymmetric cross-sectional shape and high hypotenuse-to-width ratio (HTWR). It was fabricated using basic MEMS processes, such as photolithography, anisotropic wet etching of Si, and polydimethylsiloxane (PDMS) molding. Due to the geometric similarity of a Si channel and a PDMS mold, both of which were created through the anisotropic etching process of a single crystal Si, the microfluidic channel with the asymmetric cross-sectional shape and high HTWR was easily realized. The effects of HTWR of channels on the size and uniformity of generated micro-droplets were investigated. The monodisperse micro-droplets were generated as the HTWR of the asymmetric channel was over 3.5. In addition, it was found that the flow direction of the oil solution (continuous phase) affected the size of micro-droplets due to the asymmetric channel structures. Two kinds of monodisperse droplets with different sizes were successfully generated for a wider range of flow rates using the asymmetric channel structure in the developed microfluidic device.

Slow-Off-Rate-Modified Aptamer Labeling for Fluorescence Microscopy and DNA-PAINT.

Super-resolution microscopy methods enable the visualization of biological processes on the level of a few nanometers. However, the application of these techniques in biological systems is limited by the availability of small affinity reagents. Slow off-rate-modified aptamers as nucleic acid analogues to antibodies have been successfully applied to improve the resolution and quantification of DNA-PAINT. In this chapter, we describe a protocol for using SOMAmers as labeling reagents for super-resolution microscopy.

Preliminary forensic assessment of the visualised fingerprints on nonporous substrates immersed in water using the green and optimised novel nanobio-based reagent

The discovery of forensic evidence (e.g. weapons) during forensic underwater investigations has seen an increasing trend. To date, small particle reagent (SPR) has been one of the routinely used methods for visualising fingerprints on wet, non-porous substrates. However, the long term use of SPR is detrimental to humans and environment due to the use of toxic chemicals. Although previously we have successfully developed and optimised a greener nanobio-based reagent (NBR), its suitable practical use in a more realistic scene (e.g. outdoor pond) was not evaluated. Therefore, this present research is aimed at (1) investigating the performance of NBR against the benchmark SPR in visualising fingerprints immersed in a natural outdoor pond and (2) evaluating the greenness of NBR against the analytical Eco-Scale. Results showed that the performance of the optimised NBR was mostly comparable (University of Canberra (UC) comparative scale: 0) with SPR at visualising fingerprints on three different non-porous substrates immersed in a natural outdoor pond. Observably, the NBR had higher preference towards aged fingerprints (up to 4 weeks of immersion). In addition, its greenness assessment revealed 76 points, indicating ‘excellent green analysis’. The findings gathered here further supported the practical use of the NBR in forensic investigations.

Effects of chemical warfare agent decontaminants on trace survival: Impact on fingermarks deposited on glass.

Following a chemical incident involving chemical warfare agents or more broadly, chemical weapons, there are two possible approaches in dealing with the traditional forensic analysis of contaminated exhibits. The first is to analyze the contaminated items under safe conditions (i.e. in laboratories dedicated to the handling of such substances), while the second relies on item decontamination prior to processing them in traditional forensic laboratories. One of the main limitations of the latter is the possible degradation or destruction of traces caused by the decontamination process. Hence, it is crucial to have as much information as possible on the impact of different decontamination agents and procedures on traces. This research presents experimental results on the recovery of fingermarks on glass after the application of decontaminants typically used in case of chemical incidents. The impact of 11 decontaminants on fingermarks deposited on glass and on the subsequent enhancement with cyanoacrylate and Small Particle Reagent (SPR) was evaluated (by visual examination) by four evaluators. The results of the study demonstrated that the persistence of fingermarks on glass is highly dependent on the type of decontaminant used. Decontamination agents based on the principle of nucleophilic substitution to neutralize toxic chemicals allowed good subsequent development of fingermarks with SPR. Powdered decontaminants did not show any indication of alteration of fingermarks, whereas decontamination with oxidants leads to variable results.

Review on development of wet latent fingerprints by small particle reagent

Small particle reagent technique is a method which is used to develop fingermarks on moist and smooth surfaces. It consist the theory of adherence of fine particles which is suspended in a treating solution to the oily or fatty components (water insoluble) present in in latent fingermark residue and gives a grey colored deposit. It is also considered as belonging to the family of powder dusting. The effectiveness of this technique can be increased by using fluorescent dyes in it's treating solution which can also detect faint fingermarks on multi-colored surfaces. Small particle reagent involves the use of synthetic detergent which was here replaced by saponin extracted from the fruit of Sapindus mukorossi as natural detergent. The other ingredient used in the formulation is black charcoal powder. This composition develops sharp and clear fingermarks on non-adsorbent surfaces like glass, metal sheet, plastic, laminated wood, ceramic tiles and compact discs after these were immersed in water for variable periods of time. The ability of the present formulation to detect weak chance prints not only enhances its utility, but also its potentiality in forensic investigations. The raw materials used to prepare the SPR are easily available and cost-effective.

Paper-based ELISA for fast CA 15–3 detection in point-of-care

The paper-based Enzyme-Linked Immunosorbent Assay (P-ELISA) is a promising tool for diagnostic applications because the paper matrix is characterised by low cost, short analysis time, portability, and a high surface-to-volume ratio that allows the use of a small sample and reagent volume of a few microliters. In addition, colorimetric assays are suitable for low-resource areas due to their simplicity and naked-eye detectability. Although several works have been reported using paper-based colorimetric sensors for cancer biomarkers, P-ELISA for cancer antigen 15–3 (CA 15–3) has never been reported.Thus, this work reports the development of a rapid, simple and relatively inexpensive paper-based colorimetric assay for the detection of CA 15–3 as cancer biomarker. The assay was developed on a filter paper that was previously washed and chemically oxidized with periodate to generate aldehyde functional groups on the cellulose surface. After covalent binding of the first antibody, detection was performed by a colorimetric reaction based on the oxidation of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by a peroxidase enzyme in the presence of H2O2. The colorimetric measurement was based on digital image acquisition analysed using ImageJ software. The linear range of the assay in buffer ranged 2 to 1100 U/mL. The performance of the assay was also successfully tested in human serum from Cormay®, offering a linear range from 2 to 200 U/mL. Thus, this P-ELISA sensor is suitable for the analysis of serum samples, since the physiological value in cancer patients is 30 U/mL. We believe that this proof-of-concept has the potential to be extended to be applied to other protein biomarkers and to be a suitable tool for cancer screening in developing countries.

Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line

Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent—an Affimer protein—that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.

Abstract 630: Dynamic imaging of T cell surveillance in live tumor fragments using camelid nanobodies

Abstract Immunotherapies (ITx) have revolutionized the oncology landscape. However, predicting patient responses to ITx is difficult based solely on static correlates such as TIL localization and molecular signatures. Anti-tumor immune response depends on motile surveillance by tumor infiltrating lymphocytes (TIL) which recognize antigenic determinants and engage target cells in serial stop-and-go interactions that result in cell killing. However, the hostile tumor microenvironment (TME) can cause TIL dysfunction and lack of cytotoxicity which is manifested as either suppressed or aimless TIL motility. To determine which ITx works best for a given patient, we are developing a diagnostic platform using live tumor fragments (LTF) that preserves the TME and its immune cells. Antibody-based labeling of live tissues is hampered by slow diffusion and function-altering cross-linking. To overcome these limitations, we have used small camelid-derived monovalent antibodies (nanobodies) to monitor TIL motility in LTFs. Human tumor excisions were cut into 300 x 300 x 200 µm LTFs, sorted into glass bottom multi-well plates and cultured. CD8+ cells in LTFs were labeled using an anti-hCD8a camelid VHH nanobody covalently labeled with AF594. The same reagent or a mouse anti-hCD8a whole IgG antibody was used to stain human peripheral blood mononuclear cells, and staining patterns were compared by flow cytometry. Immune responsiveness of LTFs to ITx was ascertained by flow cytometry and secreted protein assays (data shown in a companion abstract). Multiphoton microscopy revealed LTF collagen fibrils and cellular autofluorescence. A fluorescent anti-CD8a nanobody, but not a similarly labeled whole IgG, yielded good contrast and fast staining of two cell subsets. The smaller cells were 12 µm in diameter, cell surface-stained, and lacking autofluorescence, consistent with T cells. The larger cells were elongated, ramified, intracellularly stained, and distinctly autofluorescent, consistent with macrophages. 3D motility tracking revealed characteristic translational motility of the smaller cells at ~10 µm/min along collagenous structures. In contrast, larger cells exhibited only slow motility. Our results show that the motility of human CD8+ T lymphocytes can be revealed in LTF culture using a fluorescent CD8-binding camelid nanobody, likely due to its small size and monovalent binding. Further examination is needed to understand if T cell function is altered. The autofluorescence of larger, immotile cells was consistent with tumor-associated macrophages. Based on this distinction, T cells could be distinguished from the macrophages clearly. Our results support the use of camelid-derived VHH and other small monovalent reagents for live tissue lymphocyte tracking, possibly to evaluate TIL response to ITx in an LTF assay. Citation Format: Leung Kau Tang, Kelsey Tweed, Christina Scribano, David Wahl, Christin Johnson, M. Anna Zal, Jonathan Oliner, Tomasz Zal. Dynamic imaging of T cell surveillance in live tumor fragments using camelid nanobodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 630.

On-Chip Liquid Chromatography

On-chip liquid chromatography (LC) refers to LC technology that is miniaturized to fit on a microchip to enable fast, high-throughput analysis, with small sample volumes and low reagent consumption. Four different on-chip LC approaches have been developed to date: use of open-tubular, packed-particle, monolithic, and pillar array columns. These methods have been applied to proteomics as well as the analysis of small molecules and drugs in various biological samples. Recent advances in on-chip LC are summarized herein.

Neonicotinoids exposure assessment in Africanized honey bees (Apis mellifera L.) by using an environmentally-friendly sample preparation technique followed by UPLC-MS/MS

A miniaturized QuEChERS extraction method followed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed to analyze thiamethoxam and imidacloprid in 0.3 g of lyophilized Africanized honey bees (Apis mellifera L.). The work aimed to estimate honey bees' exposure to pesticides at the nanogram/gram (ng g−1) levels, using small sample and reagent quantities. Low amounts of solvents and salts were employed – 15× less than used in traditional methods. Average recoveries ranged from 64.5% to 99.7%, with repeatability below 20% for samples spiked at 3 and 167 ng g−1. LOD and LOQ were 0.7, and 3 ng g−1 for both pesticides. Applying the proposed approach, honey bee samples from different apiaries from the State of São Paulo (Brazil) were analyzed. The pesticides were detected in concentrations between 7.0 and 27.0 ng g−1. Thus, the proposed method can be used as a greener alternative to analyze the two neonicotinoids at trace levels in small quantities of bees, consequently saving chemicals and waste.

Protein Synthesis via Activated Cysteine-Directed Protein Ligation.

Proteins with a functionalized C-terminus are critical to synthesizing large proteins via expressed protein ligation. To overcome the limitations of currently available C-terminus functionalization strategies, we established an approach based on a small molecule cyanylating reagent that chemically activates a cysteine in a recombinant protein at its N-side amide for undergoing nucleophilic acyl substitution with amines. We demonstrated the versatility of this approach by successfully synthesizing RNAse H with its RNA hydrolyzing activity restored and in vitro nucleosome build with a C-terminal posttranslational modified histone H2A. This technique will expand the landscape of protein chemical synthesis and its application in new research fields significantly.

Comparative Analysis of Black Powder and Small Particle Reagent (SPR) for Development of latent Fingerprints on Non-Porous Surfaces

Comparative Analysis of Black Powder and Small Particle Reagent (SPR) for Development of latent Fingerprints on Non-Porous Surfaces

BMeS-p-A succinimidyl ester as a sulfonyl aniline dye labeling reagent

Here, we describe the synthesis and properties of BMeS-p-A-NHS as a sulfonylaniline-based fluorescent labeling reagent. Introduction of a carboxyl group into the amino group of the sulfonylaniline skeleton was achieved by the ring-opening reaction of β-propiolactone. This small labeling reagent was water-soluble, photostable, solid-state emissive, and suitable for excitation with a versatile 405-nm laser diode, emitting green fluorescence with a large Stokes shift at 527 nm. The succinimidyl ester reactive group facilitated conjugation with substrates containing amino groups.

Immersion et prises de vues photographiques des objets traités au Small Particle Reagent

While searching for fingermarks using Small Particle Reagent (SPR) on items that have been submerged, the excess SPR is usually rinsed off by spraying water, causing runoff on the treated surface or soaking in a tank of clean water before drying. However, after rinsing it is possible to immerse the object immediately in a tank of clear water in order to carry out the detection and photographic shots of fingermarks. This technique will clearly improve the contrast and the quality of the papillary pattern. Oxidation of metallic objects will also be limited, as they are not exposed to air during photographic shots.

Recovery of Latent Fingermarks on Metal Part of Motorcycle Submerged in Different Aquatic Environments

Motorcycle theft cases in Malaysia have increased quite significantly with more motorcycles were reported stolen every day. In many cases, criminals had removed the components they only required and disposed the remaining parts into various sources of water. Unfortunately, the evidence recovered underwater was thought not to have forensic value with regards to fingermark recovery. This study aimed to determine the survivability of the latent fingermarks submerged in various water environments. Latent fingermarks were deposited on metal parts of motorcycle and submerged into four aquatic environments; river, lake, mine, and tap water for two weeks with time intervals for collection of 1, 8, 24, 48, 96, 168, and 336 h. Upon recovery, the fingermark samples were developed using Small Particle Reagent (SPR). The developed fingermarks were assessed using Biometric Fingerprint Identification System (BIOFIS). The persistence of fingermarks immersed in tap water was found to last up to more than two weeks. Contrarily, fingermark in other water environments can only survive up to 96 h after being submerged. The fingermarks submerged in the lake appeared to be better preserved as compared to those submerged in the river and mine. The development of underwater fingermarks is still feasible, and identifiable fingermarks can still be recovered at the early submersion period.

Development of Natural Luminescent Powder for the Detection of Latent Fingerprint.

<b>Background and Objective:</b> Nowadays, Dyes is widely used to improve fingerprints identification test. Natural dyes are another interesting way that can be used instead of chemical dyes because of its non-toxicity and lower cost. In this research, the development of rust powder from <i>Plumeria</i> tree was applied for fingerprints identification due to its fluorescence property under UV. Rust and Small Particle Reagent (SPR), containing ZnCO<sub>3 </sub>were applied to detect hidden fingerprints on non-porous surfaces in both dried and wet condition. <b>Materials and Methods:</b> Yellowish Rust from <i>Plumeria</i> tree was extracted with ethanol, grinded, dried and then mixed with ZnCO<sub>3</sub>. Powder slurry was sprayed over fingerprint mark on different surfaces and monitored in both dried and wet condition. Visualization of fingerprint under UV was observed. Scanning microscope (SEM), UV-visible spectroscopy (UV-VIS), Fourier-Transform Infrared Spectroscopy (FTIR) and Energy-Dispersive X-ray (EDX) were also used to characterize physical and chemical properties of rust powder. <b>Results:</b> Fingerprints identification by dust technique using <i>Plumeria</i> rust powder as ingredient, provide best quality enhancement of fingerprints under UV light due to its fluorescent property, whereas a conventional technique of Small Particle Reagent technique (SPR) doesn't show fluorescent under UV. Data from SEM and FTIR show slight adhesion between zinc carbonate particles and rust powder. <b>Conclusion:</b> Fluorescence properties of rust powder is still interesting. Further improvement in powder recipe will be further investigated.

Performance of the Nanobio-Based Reagent for Visualising Wet Fingerprints Exposed to Different Levels of Water Salinity

Introduction: Recovery of evidence such as murder weapons in waterways has been commonly reported. However, visualising latent fingerprints on the immersed substrates may prove challenging due to its obliteration by physicochemical parameters (e.g. pH, temperature, salinity) as well as the mechanical effect of water. Although the use of Small Particle Reagent (SPR) for visualising latent fingerprints on wet non-porous substrates has been suggested, it contains molybdenum and titanium that are toxic, and their utilisation must be reduced. Furthermore, no study verifies the chemical and/or physical interaction between SPR and the lipid constituents of wet latent fingerprints that leads to the visualisation of ridge details. Methods: The SPR and newly developed nanobio based reagent (NBR) i.e. Candida rugosa lipase-multiwalled carbon nanotubes (CRL-MWCNTs) were used for visualising latent fingerprints on glass slides immersed in water with salinitylevels representing those of estuary and swimming pool for up to 15 days. Results: It was observed that the quality of the visualised fingerprints using NBR was comparable with that of SPR. Characterisations via instrument and bioinformatics analyses have also verified the incorporation of CRL onto MWCNTs and the chemical interaction between NBR and lipid constituents of wet latent fingerprints. Conclusions: Therefore, the newly developed NBR may prove a promising relatively greener alternative for substituting SPR for visualising latent fingerprints on glass slides immersed in different levels of water salinity.

Development of Latent Fingerprints on Non-Porous Surface with Fluorescent Dye Based Small Particle Reagent

Fingerprints are a useful source of evidence in establishing the identity of the person and hold large importance in crime scene investigations. A diverse range of physical and chemical methods are available for the development of latent fingerprints, but limited work is done on detection of prints from wet non-porous surfaces. The study aimed at developing a novel method for identification of latent fingerprints from such a type of surface. Small Particle Reagent was prepared using non-toxic chemicals and was proven to be effective in detecting latent fingerprints on Aluminum Foil. Fluorescent component, Basic Yellow-40 dye was used in the formulation. The Shelf life of the SPR solution was also considered an important factor in the study and was found to be 25 days.

One-step fast and label-free imaging array for multiplexed detection of trace avian influenza viruses

Rapid and low-cost diagnosis of multiple infectious diseases is of great significance especially in densely populated or resource-constrained settings. Herein, we developed a one-step fast and label-free imaging array for multiplexed detection of trace avian influenza virus (AIV) DNA biomarkers. By designing a series of specific and efficient catalytic hairpin assembly (CHA) amplification reactions and utilizing thioflavin T, a specific G-quadruplex fluorescence probe, three subtypes of AIV DNA biomarkers (H1N1, H7N9 and H5N1) were simultaneously and quickly detected within only 20 min, which just needed a small reagent volume of 50 μL and a smartphone instead of a spectrometer. With the combination of fluorescence imaging output and grey-level analysis, the array sensor can be on-site with the limit of detection of 136 pM, 141 pM and 129 pM for H1N1, H7N9 and H5N1, respectively. The imaging array also displayed good mismatch discrimination, excellent anti-interference, and real sample application. In view of its advantages of fast detection, low cost and multiplexed analysis, the imaging array is expected to have potential applications for early infectious disease diagnosis in resource-constrained settings.