Optical trapping and manipulation for single-particle spectroscopy and microscopy.

Optical tweezers can control the position and orientation of individual colloidal particles in solution. Such control is often desirable but challenging for single-particle spectroscopy and microscopy, especially at the nanoscale. Functional nanoparticles that are optically trapped and manipulated in a three-dimensional (3D) space can serve as freestanding nanoprobes, which provide unique prospects for sensing and mapping the surrounding environment of the nanoparticles and studying their interactions with biological systems. In this perspective, we will first describe the optical forces underlying the optical trapping and manipulation of microscopic particles, then review the combinations and applications of different spectroscopy and microscopy techniques with optical tweezers. Finally, we will discuss the challenges of performing spectroscopy and microscopy on single nanoparticles with optical tweezers, the possible routes to address these challenges, and the new opportunities that will arise.

Plasmonic SERS Biosensor Based on Multibranched Gold Nanoparticles Embedded in Polydimethylsiloxane for Quantification of Hematin in Human Erythrocytes.

This work reports a plasmonic surface-enhanced Raman scattering (SERS) biosensor that allows for quantitative analysis of hematin in erythrocytes without the need of separating it from hemoglobin (Hb). The biosensor exploits the tunable localized surface plasmon resonance (LSPR) characteristics of multibranched gold nanoparticles (M-AuNPs) and the strong plasmon coupling between an Au thin film and a flexible substrate consisting of M-AuNPs embedded in polydimethylsiloxane (PDMS) (i.e., M-AuNP-embedded PDMS substrate). In the assay, the hematin (or hematin-containing erythrocyte hemolysate) was deposited on Au film surface and covered with M-AuNP-embedded PDMS. Strong SERS signals were generated under excitation at 785 nm; the signals were sensitive to hematin concentration but not to several common coexisting biological substances. The intensities of the SERS signal (at 1623 cm-1) displayed a wide linear range using hematin concentrations in a range of at least ∼1.5 nM-1.1 µM; the limit of detection (LOD) was ∼0.03 ± 0.01 nM at a signal/noise (S/N) of 3. This assay is simple and sensitive without tedious separation procedures, thereby saving time and enhancing efficiency. This biosensor can be used to determine hematin concentration in human erythrocyte cytosols giving concentrations of ∼18.5 ± 4.5 (by averaging eight samples) and 51.5 ± 6.2 µM (by averaging three samples) for healthy and sickle erythrocytes, respectively, making it a potential application in clinical detection.

Cascade signal amplification sensing strategy for highly specific and sensitive detection of homologous microRNAs in different molecular subtypes of breast cancer.

Discriminative identification of homologous miRNAs in miRNA family with high specificity and sensitivity is crucial for accurate classification, diagnosis and prognosis of breast cancer. Herein, we report a reliable, sensitive, and selective assay by coupling fluorescence resonance energy transfer (FRET) with cascade signal amplification. The strategy is developed by designing two programmable DNA probes that can be triggered to shift from "off" to "on" state in a cascade hybridization reaction in the presence of target miRNA let-7a, leading to the generation of an amplified signal. The assay can detect concentrations as low as ∼3.0 pM let-7a and discriminate let-7a from other highly homologous members in the let-7 miRNA family. Moreover, it can also be used to determine let-7a levels at single-cell resolution and evaluate the drug efficacy of let-7a expression among various molecular types of breast cancer cell lines. The advantage of this assay is a combined result of signal generation and amplification triggered by target miRNA, which can satisfy an assay of analogous miRNA in a downregulated manner with high specificity. It has promising potential as a selective assay for homologous miRNAs in precision medicine.

Tuning the electron transport band gap of bovine serum albumin by doping with Vb12.

We report a facile and new method to tune the electron transport (ETp) band gaps of proteins (bovine serum albumin, BSA) via doping with other molecules (cyanocobalamin, Vb12). The results indicated that doping with Vb12 can enhance the ETp ability of BSA and reduce its conduction band (CB) and valence band (VB) energy levels, thereby achieving the goal of tuning protein ETp band gaps.

Raman observation of a molecular signaling pathway of apoptotic cells induced by photothermal therapy.

Plasmonic nanoparticle (NP)-mediated photothermal therapy (PPTT) has been explored as a minimally invasive approach to cancer therapy and has progressed from concept to the early stage of clinical trials. Better understanding of the cellular and molecular response to PPTT is crucial for improvement of therapy efficacy and advancement of clinical application. However, the molecular mechanism underlying PPTT-induced apoptosis is still unclear and under dispute. In this work, we used nuclear-targeting Au nanostars (Au NSs) as both a photothermal agent to specifically induce apoptosis in cancer cells and as a surface enhanced Raman spectroscopy (SERS) probe to monitor the time-dependent SERS spectra of MCF-7 cells which are undergoing apoptosis. Through SERS spectra and their synchronous and asynchronous SERS correlation maps, the occurrence and dynamics of a cascade of molecular events have been investigated, and a molecular signaling pathway of PPTT-induced apoptosis, including release of cytochrome c, protein degradation, and DNA fragmentation, was revealed, which was also demonstrated by metabolomics, agarose gel electrophoresis, and western blot analysis, respectively. These results indicated that PPTT-induced apoptosis undergoes an intrinsic mitochondria-mediated apoptosis pathway. Combined with western blot results, this intrinsic mitochondria-mediated apoptosis pathway was further demonstrated to be initiated by a BH3-only protein, BID. This work is beneficial for not only improving the fundamental understanding of the molecular mechanism of apoptosis induced by PPTT but also for guiding the modulation of PPTT to drive forward its clinical application.

Synthesis of Nitrogen-Doped Graphene Quantum Dots at Low Temperature for Electrochemical Sensing Trinitrotoluene.

Nitrogen-doped graphene quantum dots (N-GQDs) are synthesized at low temperature as a new catalyst allowing electrochemical detection of 2,4,6-trinitrotoluene (TNT). N-GQDs are made by an oxidative ultrasonication of graphene oxide (GO) forming nanometer-sized species, which are then chemically reduced and nitrogen doped by reacting with hydrazine. The as-synthesized N-GQDs have an average diameter of ∼2.5 nm with an N/C atomic ratio of up to ∼6.4%. To detect TNT, TNT is first accumulated on N-GQDs modified glassy carbon (N-GQDs/GC) electrode by holding the electrode at a 0 V versus Ag/AgCl for 150 s in an aqueous TNT solution. Next, the N-GQDs/GC electrode with accumulated TNT is transferred to a fresh PBS solution (0.1 M, pH 7.0, without TNT), where the TNT reduction current at -0.36 V versus Ag/AgCl in a linear scan voltammogram (LSV) shows a linear response to TNT concentration in the aqueous solution from 1 to 400 ppb, with a correlation coefficient of 0.999, a detection limit of 0.2 ppb at a signal/noise (S/N) of 3, and a detection sensitivity of 363 ± 7 mA mM(-1) cm(-2). The detection limit of 0.2 ppb of TNT for this new method is much lower than 2 ppb set by the U.S. Environmental Protection Agency for drinking water. Therefore, N-GQDs allow an electrochemical method for assaying TNT in drinking water to determine if levels of TNT are safe or not.

Highly sensitive methyltransferase activity assay and inhibitor screening based on fluorescence quenching of graphene oxide integrated with the site-specific cleavage of restriction endonuclease.

We report a sensitive and selective approach for the DNA methyltransferase (MTase) activity assay and MTase inhibitor screening by coupling the fluorescence quenching of graphene oxide with site-specific cleavage of a restriction endonuclease.

Enhancing the electrochemical reduction of hydrogen peroxide based on nitrogen-doped graphene for measurement of its releasing process from living cells.

An electrochemical approach for measuring the dynamic process of H(2)O(2) (a major ROS) release from living cells is reported. This approach, which is based on enhanced reduction of H(2)O(2) by nitrogen-doped graphene, could be potentially useful in the study of downstream biological effects of various stimuli in physiology and pathology.

Electrochemical measurement of the flux of hydrogen peroxide releasing from RAW 264.7 macrophage cells based on enzyme-attapulgite clay nanohybrids.

Determination of cellular ROS (reactive oxygen species) could lead to a better understanding of the clinical consequences of the enhancement in ROS concentration, and assisting in studies of the biological effect of ROS in cells. This work developed an electrochemical approach for measuring the flux of H(2)O(2) (a major ROS in living organisms) releasing from RAW 264.7 macrophage cells. This approach is based on the electrocatalytic reduction of the releasing H(2)O(2) at the biosensor of HRP-attapulgite/GC, which was fabricated by depositing the horseradish peroxidase-attapulgite nanohybrids on the glassy carbon (GC) electrode. The biosensor exhibited a rapid response, a wide linear range, a high sensitivity, a low detection limit, as well as good stability and repeatability due to using the natural mineral (attapulgite) as the enzyme immobilization substrate. In addition, some common coexisting ROS and compounds in biological system such as hypochlorite (OCl(-)), nitric oxide (NO), peroxynitrite (ONOO(-)), and ascorbic acid (AA) etc., did not cause any interference due to the use of a low operating potential (-400mV, versus SCE). Moreover, the developed approach can also be used for studying the effects of the stimulator loading and a variety of stimuli on the generation of H(2)O(2) in cells and the release flux of H(2)O(2) from cells. Therefore, this work has demonstrated a simple and effective sensing platform for detection of cellular H(2)O(2) released from cells such as RAW 264.7 cells, which has potential utility to cellular biology and pathophysiology.