Rapid highly sensitive general protein quantification through on-chip chemiluminescence.

Protein detection and quantification is a routinely performed procedure in research laboratories, predominantly executed either by spectroscopy-based measurements, such as NanoDrop, or by colorimetric assays. The detection limits of such assays, however, are limited to μ M concentrations. To establish an approach that achieves general protein detection at an enhanced sensitivity and without necessitating the requirement for signal amplification steps or a multicomponent detection system, here, we established a chemiluminescence-based protein detection assay. Our assay specifically targeted primary amines in proteins, which permitted characterization of any protein sample and, moreover, its latent nature eliminated the requirement for washing steps providing a simple route to implementation. Additionally, the use of a chemiluminescence-based readout ensured that the assay could be operated in an excitation source-free manner, which did not only permit an enhanced sensitivity due to a reduced background signal but also allowed for the use of a very simple optical setup comprising only an objective and a detection element. Using this assay, we demonstrated quantitative protein detection over a concentration range of five orders of magnitude and down to a high sensitivity of 10 pg mL - 1 , corresponding to pM concentrations. The capability of the platform presented here to achieve a high detection sensitivity without the requirement for a multistep operation or a multicomponent optical system sets the basis for a simple yet universal and sensitive protein detection strategy.Engineering and Physical Sciences Research Council Schmidt Science Fellows program in partnership with the Rhodes Trust European Research Council Newman Foundatio

Microfluidic Techniques for Protein Detection and Manipulation

Protein-protein interactions are of central importance in biological systems. The interplay between proteins determines the functioning of such systems. Accordingly, a variety of methods have been established for the study of protein-protein interactions, but each class of methods has key limitations. In particular, dissociation constants of biologically relevant interactions are often in the nM to μM range, probing at such low concentrations requires highly sensitive techniques. An improved understanding of the functioning of proteins lead to increased drug usage in combating diseases. Bio-compatible and bio-degradable drug-carrying vehicles are highly desirable. This thesis describes the unique properties of fluids when confined to small length scales, which enable analysis of proteins at low concentrations and manipulation of protein assembly for the manufacture of function materials. The analysis on protein-protein interactions evolved around the differences in hydrodynamic radii of proteins in the monomer and bound states, which change the diffusion constants of the protein. By employing a non-specific label, the rate of diffusion of various proteins can be detected. An existing microfluidic platform was initially employed to investigate biologically important interactions, yet detection limit and precision still posed as problems hindering measurements. A versatile, universally-applicable chemiluminescent platform for protein detection was introduced in this thesis. The intrinsically low background noise from chemiluminescence provides a low detection limit where tight-binding interactions or low concentration samples can be easily analysed. Lastly, microdroplets fabricated using two immiscible aqueous systems offer high bio-compatibility, and thus, when cross-linked with protein fibrils, would serve as optimal vehicles for drug delivery or cell encapsulation purposes

Real-Time Highly-Sensitive Protein Quantification Through On-Chip Chemiluminescence

A range of experimental methods have been developed to achieve highly sensitive detection and quantification of proteins. The majority of these methods rely on fluorescence-mediated readouts and, as such, their sensitivity can be affected by factors such as photobleaching of fluorophores and background signal from the illumination source. Both of these limitations can be overcome by using chemiluminescence-based detection: in contrast to fluorescence, chemiluminescence can be generated in an excitation source free manner, which allows for a significant reduction in background noise and for the use of an optical setup that comprises only a detection element. Here, we develop a highly-sensitive protein quantification platform by combining chemiluminescent detection of proteins with microfluidic mixing and detection. We use the platform to demonstrate quantitative detection of proteins over a concentration range of five orders of magnitude and down to 10 pg mL−1, corresponding to pM concentrations. Owing to the general presence of amine groups in peptides and proteins, our demonstrated system is applicable to characterising any protein sample and it can be used to quantify unlabelled samples. </div

Comparative Analysis of Chloroplast Genomes of Dalbergia Species for Identification and Phylogenetic Analysis

Dalbergia L.f. is a pantropical genus consisting of 269 species of trees, shrubs, and woody lianas. This genus is listed in CITES Appendices because of illegal logging and trafficking driven by the high economic value of its heartwood. Some species are also used medicinally. Species identification of Dalbergia timber and herbs is challenging but essential for CITES implementation. Molecular methods had been developed for some timber species, mostly from Madagascar and Southeast Asia, but medicinal species in south China were usually not included in those studies. Here, we sequenced and assembled the chloroplast genomes of five Dalbergia species native to Hong Kong, four of which are medicinal plants. Our aim is to find potential genetic markers for the identification of medicinal Dalbergia species based on divergence hotspots detected in chloroplast genomes after comparative and phylogenetic analysis. Dalbergia chloroplast genomes displayed the typical quadripartite structure, with the 50 kb inversion found in most Papilionoideae lineages. Their sizes and gene content are well conserved. Phylogenetic tree of Dalbergia chloroplast genomes showed an overall topology similar to that of ITS sequences. Four divergence hotspots (trnL(UAA)-trnT(UGU), ndhG-ndhI, ycf1a and ycf1b) were identified and candidate markers for identification of several Dalbergia species were suggested

The Complete Chloroplast Genomes of Nine Smilacaceae Species from Hong Kong: Inferring Infra- and Inter-Familial Phylogeny

The Smilacaceae is a cosmopolitan family consisting of 200–370 described species. The family includes two widely accepted genera, namely Smilax and Heterosmilax. Among them, the taxonomical status of Heterosmilax has been continuously challenged. Seven Smilax and two Heterosmilax species can be found in Hong Kong, with most of them having medicinal importance. This study aims to revisit the infra-familial and inter-familial relationships of the Smilacaceae using complete chloroplast genomes. The chloroplast genomes of the nine Smilacaceae species from Hong Kong were assembled and annotated, which had sizes of 157,885 bp to 159,007 bp; each of them was identically annotated for 132 genes, including 86 protein-coding genes, 38 transfer RNA genes, and 8 ribosomal RNA genes. The generic status of Heterosmilax was not supported because it was nested within the Smilax clade in the phylogenetic trees, echoing previous molecular and morphological studies. We suggest delimitating the genus Heterosmilax as a section under the genus Smilax. The results of phylogenomic analysis support the monophyly of Smilacaceae and the exclusion of Ripogonum from the family. This study contributes to the systematics and taxonomy of monocotyledons, authentication of medicinal Smilacaceae, and conservation of plant diversity