Plasmonic tweezers for optical manipulation and biomedical applications

Hongtao Tan, Huiqian Hu, Lin Huang and Kun Qian

Plasmonic tweezers are an emerging research topic because of their breakthrough in the conventional diffraction limit and precise manipulation at the nanoscale. Notably, their compatibility with analytical techniques (e.g. fluorescence, surface-enhanced Raman scattering (SERS), and laser desorption/ionization mass spectrometry (LDI MS)) opens up opportunities in optical manipulation and biomedical applications. Herein, we first introduce the structures and trapping forces, followed by a summary of the properties of plasmonic tweezers. The optical trapping of biosamples by plasmonic tweezers are then reviewed, including microorganisms and biomolecules. Finally, we highlight the integration of plasmonic tweezers with analytical techniques towards bioanalytical applications. We conclude with perspectives on the future directions for this topic. We foresee the upcoming era of biological detection by plasmonic tweezing in both academy and industry, which calls for the interest and efforts of scientists from diverse fields.

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True one cell chemical analysis: a review

Imesha W. De Silva, Amanda R. Kretsch, Holly-May Lewis, Melanie Bailey and Guido F. Verbeck

The constantly growing field of True One Cell (TOC) analysis has provided important information on the direct chemical composition of various cells and cellular components. Since the heterogeneity of individual cells has been established, more researchers are interested in the chemical differences between individual cells; TOC is the only form of analysis that can provide this information. This has resulted in the constant development of new technologies and methods. This review highlights the common techniques for micro- and nanomanipulation, Raman spectroscopy, microscopy, and mass spectrometric imaging as they pertain to TOC chemical analysis.

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Single-cell analysis reveals the effects of glutaraldehyde and formaldehyde on individual Nosema bombycis spores

Zhenbin Miao, Pengfei Zhang, Yu Zhang, Xuhua Huang, Junxian Liu and Guiwen Wang

Nosema bombycis (Nb) is the pathogen that causes pebrine in silkworms. Aldehydes are effective disinfectants commonly used in sericulture. However, the precise mechanism of their action on Nb spores remains unclear. Here, we used laser tweezers Raman spectroscopy to investigate the effects of glutaraldehyde and formaldehyde on individual Nb spores, as well as phase contrast microscopy imaging to monitor the germination dynamics of individual treated spores, to acquire a deeper understanding of the mechanism of action of aldehydes and to provide a theoretical reference for establishing an effective strategy for disease control in sericulture. The positions of the Raman peaks remained constant during treatment. The Raman intensity was enhanced and the germination rate of the spores significantly decreased with treatment time. Tlag, the time when individual spores begin to germinate, and Tgerm, the time for complete germination, increased with enhanced treatment. The germination time (ΔTgerm) showed no significant difference from that for untreated spores. Heterogeneity was shown, which is relevant to the resistance of Nb spores to aldehydes. The results indicate that glutaraldehyde and formaldehyde do not destroy the spore wall and plasma membrane, do not cause the leakage of intracellular components, and might not damage the extrusion apparatus. The effects of aldehydes on Nb spores are mainly on the spore coat. They may block the external factors that stimulate spore germination. Single-cell analysis based on novel optical techniques reveals the action of chemical sporicides on microsporidia spores in real time and explains the heterogeneity of cell stress resistance. These applications of new techniques offer new insight into traditional disinfectants.

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A review of sorting, separation and isolation of cells and microbeads for biomedical applications: microfluidic approaches

Arash Dalili, Ehsan Samiei and Mina Hoorfar

Several biomedical analyses are performed on particular types of cells present in body samples or using functionalized microparticles. Success in such analyses depends on the ability to separate or isolate the target cells or microparticles from the rest of the sample. In conventional procedures, multiple pieces of equipment, such as centrifuges, magnets, and macroscale filters, are used for such purposes, which are time-consuming, associated with human error, and require several operational steps. In the past two decades, there has been a tendency to develop microfluidic techniques, so-called lab-on-a-chip, to miniaturize and automate these procedures. The processes used for the separation and isolation of the cells and microparticles are scaled down into a small microfluidic chip, requiring very small amounts of sample. Differences in the physical and biological properties of the target cells from the other components present in the sample are the key to the development of such microfluidic techniques. These techniques are categorized as filtration-, hydrodynamic-, dielectrophoretic-, acoustic- and magnetic-based methods. Here we review the microfluidic techniques developed for sorting, separation, and isolation of cells and microparticles for biomedical applications. The mechanisms behind such techniques are thoroughly explained and the applications in which these techniques have been adopted are reviewed.

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Single-cell assay on microfluidic devices

Qiushi Huang, Sifeng Mao, Mashooq Khan and Jin-Ming Lin

Advances in microfluidic techniques have prompted researchers to study the inherent heterogeneity of single cells in cell populations. This would be helpful in the identification of major diseases and the design of personalized medicine. Different microfluidic approaches provide a variety of functions in the process of single-cell analysis. In this review, we take a broad overview of various microfluidic-based approaches for single-cell isolation, single-cell lysis, and single-cell analysis. Up-to-date flagship techniques and the pros and cons of these methods are discussed in detail.

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Optical guiding-based cell focusing for Raman flow cell cytometer

Ravi Shanker Verma, Sunita Ahlawat and Abha Uppal

We report the use of an optical guiding arrangement generated in a microfluidic channel to produce a stream of single cells in a line for single-cell Raman spectroscopic analysis. The optical guiding arrangement consisted of dual-line optical tweezers, generated using a 1064 nm laser, aligned in the shape of a ‘Image ID:c8an00037a-u1.gif’ symbol. By controlling the laser power in the tweezers and the flow rate in the microfluidic channel, a single line flow of cells could be produced in the tail of the guiding arrangement, where the 514.5 nm Raman excitation beam was also located. Furthermore, by resonantly exciting the Raman spectrum, a good-quality Raman spectrum could be recorded from the flowing single cells as they passed through the Raman excitation focal spot without the need to trap the cells. As a proof of concept, it was shown that red blood cells (RBCs) could be guided to the tail of the optical guide and the Raman spectra of the resonantly excited cells could be recorded in a continuous manner without trapping the cells at a cell flow rate of ∼500 cells per h. From the recorded spectra, we were able to distinguish between RBCs containing hemoglobin in the normal form (normal-RBCs) and the met form (met-RBCs) from a mixture of RBCs comprising met-RBCs and normal-RBCs in a ratio of 1 : 9.

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Recent advances in the use of microfluidic technologies for single cell analysis

Travis W. Murphy, Qiang Zhang, Lynette B. Naler, Sai Ma and Chang Lu

The inherent heterogeneity in cell populations has become of great interest and importance as analytical techniques have improved over the past decades. With the advent of personalized medicine, understanding the impact of this heterogeneity has become an important challenge for the research community. Many different microfluidic approaches with varying levels of throughput and resolution exist to study single cell activity. In this review, we take a broad view of the recent microfluidic developments in single cell analysis based on microwell, microchamber, and droplet platforms. We cover physical, chemical, and molecular biology approaches for cellular and molecular analysis including newly emerging genome-wide analysis.

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Measurement of pH-dependent surface-enhanced hyper-Raman scattering at desired positions on yeast cells via optical trapping

Yasutaka Kitahama, Hiroaki Hayashi, Tamitake Itoh and Yukihiro Ozaki

Surface-enhanced hyper-Raman scattering (SEHRS) spectra were obtained at desired positions on yeast by focusing a continuous wave near-infrared laser beam while silver nanoparticles (AgNPs) were simultaneously optically trapped. However, the optically trapped colloidal AgNP suspension bubbled up at the focusing point, preventing spectral measurement. In the case of optically trapped AgNPs functionalized with 4-mercaptobenzoic acid (p-MBA), surface-enhanced hyper-Rayleigh scattering was considerably strong, indicating the suppression of the photothermal conversion to form the bubble. Interestingly, the SEHRS peaks that are attributed not only to p-MBA, but also to other species, were very occasionally observed. They may be partly assigned to the β1,3 glucan and protein amide II band. The SEHRS peak at 1366 cm−1 was barely visible in the measurements of conventional baker's yeast even in the suspension (pH 9) despite the effects of high pH on p-MBA. In contrast, the SEHRS peak in the measurements of yeast for biological applications was occasionally observed at 1366 cm−1. This suggests that acidity is correlated with fermentation efficiency. At different positions on single yeast cells, the intensity of the SEHRS peak at 1366 cm−1 varied. This result represents the pH distribution on yeast.

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In vivo quantitative Raman-pH sensor of arterial blood based on laser trapping of erythrocytes

Man man Lin, Bin Xu, huilu yao, Aiguo Shen and Jiming Hu

We report on a continuous and non-invasive approach in vivo to monitor arterial blood pH based on laser trapping and Raman detection of single live erythrocytes. A home-built confocal laser tweezers Raman system (LTRS) is applied to trace the live erythrocytes under different pH values of extracellular environment to record their corresponding Raman changes in vitro and in vivo. The analysis results in vitro show that when the extracellular environment pH changes from 6.5 to 9.0, two Raman intensity ratio (R1603, 1616=I1603/I1616) of single erythrocytes decreases regularly, what is more, there has a good linear relationship between these two variables, and the linearity is 0.985, which is also verified successfully via in vivo Raman measurements. These results demonstrate that the Raman signal of single live erythrocytes is possible as a marker of extracellular pH value. This in vivo and quantitative Raman-pH sensor of arterial blood will be an important candidate for monitoring the acid-base status during treatment of ill patients and some major surgery because of its continuous and non-invasive characters.

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Use of Raman optical tweezers for cell cycle analysis

Sunita Ahlawat, Aniket Chowdhury, Abha Uppal, Nitin Kumar and Pradeep Kumar Gupta

We report the results of our investigations on the use of Raman optical tweezers for label free analysis of cells in different phases of their cell cycle. The studies performed on human colon adenocarcinoma (Colo-205) cells synchronized in G0/G1 and G2/M phases showed that the DNA Raman band at 783 cm-1 in the Raman spectra of optically trapped cells can provide information about the DNA content in the nucleus of the cell without the need for isolation of nucleus. The histograms of intensity of this band among the cell populations were found to corroborate the results obtained from fluorescence image cytometry performed on DAPI stained cells.

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Automated analysis of single cells using Laser Tweezers Raman Spectroscopy

Stephen Casabella, Peter Gardner, Patricia Scully and N J Goddard

In recent years, significant progress has been made into the label-free detection and discrimination of individual cancer cells using Laser Tweezers Raman Spectroscopy (LTRS). However, the majority of examples reported have involved manual trapping of cells, which is time consuming and may lead to different cell lines being analysed in discrete batches. A simple, low-cost microfluidic flow chamber is introduced which allows single cells to be optically trapped and analysed in an automated fashion, greatly reducing the level of operator input required. Two implementations of the flow chamber are discussed here; a basic single-channel device in which the fluid velocity is controlled manually, and a dual-channel device which permits the automated capture and analysis of multiple cell lines with no operator input. Results are presented for the discrimination of live epithelial prostate cells and lymphocytes, together with a consideration of the consequences of traditional ‘batch analysis’ typically used for LTRS of live cells.

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Towards high-throughput microfluidic Raman-activated cell sorting

Qiang Zhang, Peiran Zhang, Honglei Gou, Chunbo Mou, Wei E. Huang, Menglong Yang, Jian Xu and Bo Ma

Raman-activated cell sorting (RACS) is a promising single-cell analysis technology that is able to identify and isolate individual cells of targeted type, state or environment from an isogenic population or complex consortium of cells, in a label-free and non-invasive manner. However, compared with those widely used yet labeling-required or staining-dependent cell sorting technologies such as FACS and MACS, the weak Raman signal greatly limits the further development of the existing RACS systems to achieve higher throughput. Strategies that can tackle this bottleneck include, first, improvement of Raman-acquisition efficiency and quality based on advanced Raman spectrometers and enhanced Raman techniques; second, development of novel microfluidic devices for cell sorting followed by integration into a complete RACS system. Exploiting these strategies, prototypes for a new generation of RACS have been demonstrated, such as flow-based OT-RACS, DEP-RACS, and SERS/CARS flow cytometry. Such high-throughput microfluidic RACS can provide biologists with a powerful single-cell analysis tool to explore the scientific questions or applications that have been beyond the reach of FACS and MACS.

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Controlled translocation of DNA through nanopores in carbon nano-, silicon-nitride- and lipid-coated membranes

Andy Sischka, Lukas Galla, Andreas J. Meyer, Andre Spiering, Sebastian Knust, Michael Mayer, Adam R. Hall, André Beyer, Peter Reimann, Armin Gölzhäuser and Dario Anselmetti

We investigated experimentally and theoretically the translocation forces when a charged polymer is threaded through a solid-state nanopore and found distinct dependencies on the nanopore diameter as well as on the nano membrane material chemistry. For this purpose we utilized dedicated optical tweezers force mechanics capable of probing the insertion of negatively charged double-stranded DNA inside a helium-ion drilled nanopore. We found that both the diameter of the nanopore and the membrane material itself have significant influences on the electroosmotic flow through the nanopore and thus on the threading force. Compared to a bare silicon-nitride membrane, the threading of DNA through only 3 nm thin carbon nano membranes as well as lipid bilayer-coated nanopores increased the threading force by 15% or 85%, respectively. This finding was quantitatively described by our recently developed theoretical model that also incorporates hydrodynamic slip effects on the translocating DNA molecule and the force dependence on the membrane thickness. The additional measurements presented in this paper further support our model.

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Composite SERS-based satellites navigated by optical tweezers for single cell analysis

Inna Stetciura, Alexey Yashchenok, Admir Masic, Evgeny Lyubin, Olga Inozemtseva, Maria Drozdova, Elena Markvichova, Boris N. Khlebtsov, Andrey A. Fedyanin, Gleb Sukhorukov , Dmitry Alexandrovich Gorin and Dmitry Volodkin

Here we have designed composite SERS active micro-satellites possessing a dual role working as: i) effective probes for cellular composition and ii) optically movable and easily detectable markers. The satellites were synthesized by the layer-by-layer assisted decoration of silica microparticles with metal (gold or silver) nanoparticles and astralen in order to ensure satellite SERS-based microenvironment probing and satellite recognition, respectively. A combination of optical tweezers and Raman spectroscopy can be used to navigate the satellites to a certain cellular compartment followed by the cellular uptake and also to probe the intracellular composition. The developed approach may serve in future as a tool for the single cell analysis focusing on both extracellular and intracellular studies with nanometer precision due to the multilayer surface design.

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Microfluidics for Research and Applications in Oncology

Parthiv Chaudhuri, Majid Ebrahimi Warkiani, Tengyang Jing, Kenry Kenry and Chwee Teck Lim

Cancer is currently one of the top non-communicable human diseases and continual research and developmental efforts are being made to better understand and manage this disease. More recently, with improved understanding in cancer biology as well as advancement made in microtechnology and rapid prototyping, microfluidics is increasingly being explored and even validated for use in the detection, diagnosis and treatment of cancer. With inherent advantages such as small sample volume, high sensitivity and fast processing time, microfluidics is well-positioned to serve as a promising platform for applications in oncology. In this review, we look at recent advances in the use of microfluidics - from basic research such as understanding cancer cell phenotypes as well as metastatic behaviors to applications such as detection, diagnosis, prognosis and drug screening. We then conclude with a future outlook on this promising technology.

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Molecular Weight Characterization of Globular Proteins using Optical Nanotweezers

Skyler Wheaton and Reuven Gordon

We trap a set of molecular weight standard globular proteins using a double nanohole optical trap. The root mean squared variation of the trapping laser transmission intensity gives a linear dependence with the molecular weight, showing the potential for analysis of globular proteins. The characteristic time of the autocorrelation of the trapping laser intensity variations scales with a -2/3 power dependence with the volume of the particle. A hydrodynamic laser tweezer model is used to explain these dependencies. Since this is a single particle technique that operates in solution and can be used to isolate an individual particle, we believe that it provides an interesting alternative to existing analysis methods and shows promise to expand the capabilities of protein related studies to the single particle level.

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Label-Free Free-Solution Nanoaperture Optical Tweezers for Single Molecule Protein Studies

Ahmed A Al Balushi, Abhay Kotnala, Skyler Wheaton, Ryan M. Gelfand, Yashaswini Rajashekara and Reuven Gordon

Nanoaperture optical tweezers are emerging as useful label-free, free-solution tools for the detection and identification of biological molecules and their interactions at the single molecule level. Nanoaperture optical tweezers provide a low-cost, scalable, straight-forward, high-speed and highly sensitive (SNR ~ 33) platform to observe real-time dynamics and to quantify binding kinetics of protein-small molecule interactions without the need to use tethers or labeling. Such nanoaperture-based optical tweezers, which are 1000 times more efficient than conventional optical tweezers, have been used trap and isolate single DNA molecules and to study proteins like p53, which has been claimed to be in mutant form for 75% of human cancers. More recently, nanoaperture optical tweezers have been used to probe the low-frequency (in the single digit wavenumber range) Raman active modes of single nanoparticles and proteins. Here we review recent developments in the field of nanoaperture optical tweezers and how they have been applied to protein-antibody interactions, protein – small molecule interactions including single molecule binding kinetics, and protein-DNA interactions. In addition, recent works on the integration of nanoaperture optical tweezers at the tip of optical fiber and in microfluidic environment are presented.

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Squeezing red blood cells on an optical waveguide to monitor cell deformability during blood storage

Balpreet Singh Ahluwalia, Peter McCourt, Ana Oteiza, James S. Wilkinson, Thomas R Huser and Olav Gauta Helleso 

Red blood cells squeeze through micro-capillaries as part of blood circulation in the body. The deformability of red blood cells is thus critical for blood circulation. In this work, we report a method to optically squeeze red blood cells using the evanescent field present on top of a planar waveguide chip. The optical forces from a narrow waveguide are used to squeeze red blood cells to a size comparable to the waveguide width. Optical forces and pressure distributions on the cells are numerically computed to explain the squeezing process. The proposed technique is used to quantify the loss of blood deformability that occurs during blood storage lesion. Squeezing red blood cells using waveguides is a sensitive technique and works simultaneously on several cells, making the method suitable for monitoring stored blood.

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The Grab-and-Drop Protocol: A Novel Strategy for Membrane Protein Isolation and Reconstitution from Single Cells

Angelika Schrems, John Phillips, Duncan Robert Casey, Douglas Wylie, Miroslava Novakova, Uwe B Sleytr, David Klug, Mark A A Neil, Bernhard Schuster and Oscar Ces

We present a rapid and robust technique for the sampling of membrane-associated proteins from the surface of a single, live cell and their subsequent deposition onto a solid-supported lipid bilayer. As a proof of principle, this method has been used to extract green fluorescent protein (EGFP) labelled K-ras proteins located at the inner leaflet of the plasma membrane of colon carcinoma cells and to transfer them to an S-layer supported lipid bilayer system. The technique is non-destructive, meaning that both the cell and proteins are intact after the sampling operation, offering the potential for repeated measurements of the same cell of interest. This system provides the ideal tool for the investigation of cellular heterogeneity, as well as a platform for the investigation of rare cell types such as circulating tumour cells.

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Absolute Quantification of Protein Copy Number using a Single-Molecule-Sensitive Microarray

Edward Burgin, Ali Salehi-Reyhani, Michael Barclay, Aidan Thomas Brown, Joseph Kaplinsky, Miroslava Novakova, Mark A A Neil, Oscar Ces, Keith R Willison and David Klug

We report the use of a microfluidic microarray incorporating single molecule detection for the absolute quantification of protein copy number in solution. In this paper we demonstrate protocols which enable calibration free detection for two protein detection assays. An EGFP protein assay has a limit of detection of <30 EGFP proteins in a microfluidic analysis chamber (limited by non-specific background binding), with a measured limit of linearity of approximately 6×106 molecules of analyte in the analysis chamber and a dynamic range of >5 orders of magnitude in protein concentration. An antibody sandwich assay was used to detect unlabelled human tumour suppressor protein p53 with a limit of detection of approximately 21 p53 proteins and a dynamic range of >3 orders of magnitude. We show that these protocols can be used to determine the absolute protein copy number at the single cell level in two human cancer cell lines.

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