Target trapping and in situ single-cell genetic marker detection with a focused optical beam

Hengji Cong, Fong-Chuen Loo, Jiajie Chen, Yuye Wang, Siu-Kai Kong, Ho-Pui Ho

Optical trapping of single particles or cells with the capability of in situ bio-sensing or genetic profiling opens the possibility of rapid screening of biological specimens. However, common optical tweezers suffer from the lack of long-range forces. Consequently, their application areas are predominantly limited to target manipulation instead of biological diagnostics. To solve this problem, we herein report an all-in-one approach by combining optical forces and convective drag forces generated through localized optothermal effect for long-range target manipulation. The device consists of a 2D array of gold coated polydimethylsiloxane (PDMS) micro-wells, which are immersed by colloidal particles or cell solution. Upon excitation of a 785-nm laser, the hydrodynamic convective force and optical forces will drag the targets of interest into their designated micro-wells. Moreover, the plasmonic thermal dissipation provides a constant temperature environment for following cell analysis procedures of cell isolation, lysis and isothermal nucleic acid amplification for the detection of genetic markers. With the merits of fabrication simplicity, short sample-to-answer cycle time and the compatibility with optical microscopes, the reported technique offers an attractive and highly versatile approach for on-site single cell analysis systems.

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Spectral-optical-tweezer-assisted fluorescence multiplexing system for QDs-encoded bead-array bioassay

Qinghua He, Xuejing Chen, Yonghong He, Tian Guan, Guangxia Feng, Bangrong Lu, Bei Wang, Xuesi Zhou, Liangshan Hu, Donglin Cao

As an efficient tool in the multiplexed detection of biomolecules, bead-array could achieve separation-free detection to multiple targets, making it suitable to analyze valuable and scarce samples like antigen and antibody from living organism. Herein, we propose a spectral-optical-tweezer-assisted fluorescence multiplexing system to analyze biomolecule-conjugated bead-array. Using optical tweezer, we trapped and locked beads at the focus to accept stimulation, offering a stable and optimized analysis condition. Moving the system focus and scanning the sample slide, we achieved emissions collection to QDs-encoded bead-array after the multiplexed detection. The emission spectra of fluorescence were collected and recorded by the spectrometer. By recognizing locations of decoding peaks and counting the intensities of label signals of emission spectra, we achieved qualitative and quantitative detection to targets. As proof-of-concept studies, we use this system to carry out multiplexed detection to various types of anti-IgG in the single sample and the detection limit reaches 1.52 pM with a linear range from 0.31 to 10 nM. Through further optimization of experimental conditions, we achieved specific detection to target IgG with sandwich method in human serum and the detection limit reaches as low as 0.23 pM with a linear range from 0.88 to 28 pM, validating the practical application of this method in real samples.

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A force sensor that converts fluorescence signal into force measurement utilizing short looped DNA

Golam Mustafa, Cho-Ying Chuang, William A. Roy, Mohamed M.F arhath, Nilisha Pokhrel, Yue Ma, Kazuo Nagasawa, Edwin Antony, Matthew J. Comstock, Soumitra Basu, Hamza Balci

A force sensor concept is presented where fluorescence signal is converted into force information via single-molecule Förster resonance energy transfer (smFRET). The basic design of the sensor is a ~100 base pair (bp) long double stranded DNA (dsDNA) that is restricted to a looped conformation by a nucleic acid secondary structure (NAS) that bridges its ends. The looped dsDNA generates a tension across the NAS and unfolds it when the tension is high enough. The FRET efficiency between donor and acceptor (D&A) fluorophores placed across the NAS reports on its folding state. Three dsDNA constructs with different lengths were bridged by a DNA hairpin and KCl was titrated to change the applied force. After these proof-of-principle measurements, one of the dsDNA constructs was used to maintain the G-quadruplex (GQ) construct formed by thrombin binding aptamer (TBA) under tension while it interacted with a destabilizing protein and stabilizing small molecule. The force required to unfold TBA-GQ was independently investigated with high-resolution optical tweezers (OT) measurements that established the relevant force to be a few pN, which is consistent with the force generated by the looped dsDNA. The proposed method is particularly promising as it enables studying NAS, protein, and small molecule interactions using a highly-parallel FRET-based assay while the NAS is kept under an approximately constant force.

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One-step separation-free detection of carcinoembryonic antigen in whole serum: Combination of two-photon excitation fluorescence and optical trapping

Cheng-Yu Li, Di Cao, Chu-Bo Qi, Hong-Lei Chen, Ya-Tao Wan, Yi Lin, Zhi-Ling Zhang, Dai-Wen Pang, Hong-Wu Tang

Direct analysis of biomolecules in complex biological samples remains a major challenge for fluorescence-based approaches due to the interference of background signals. Herein, we report an analytical methodology by exploiting a single low-cost near-infrared sub-nanosecond pulse laser to synchronously actualize optical trapping and two-photon excitation fluorescence for senstive detection of carcinoembryonic antigen (CEA) in buffer solution and human whole serum with no separation steps. The assay is performed by simultaneously trapping and exciting the same immune-conjugated microsphere fabricated with a sandwich immunization strategy. Since the signal is strictly limited in the region of a three-dimensional focal volume where the microsphere is trapped, no obvious background signal is found to contribute the detected signals and thus high signal-to-background data are obtained. As a proof-of-concept study, the constructed platform exhibits good specificity for CEA and the detection limit reaches as low as 8 pg/mL (45 fM) with a wide linear range from 0.01 to 60 ng/mL in the both cases. To investigate the potential application of this platform in clinical diagnosis, 15 cases of serum samples were analyzed with satisfactory results, which further confirm the applicability of this method.

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Dual-component gene detection for H7N9 virus – the combination of optical trapping and bead-based fluorescence assay

Di Cao, Cheng-Yu Li, Ya-Feng Kang, Yi Lin, Ran Cui, Dai-Wen Pang, Hong-Wu Tang

We present a strategy of dual-component gene detection for avian influenza A virus H7N9 by combining optical trapping and bead-based fluorescence bioassays. A low-cost 473 nm continuous DPSS laser, polystyrene (PS) beads with two different sizes (3 µm and 5 µm in diameter) and streptavidin-modified 605 nm quantum dots (SA-QDs) were exploited in this platform. The beads were employed to enrich the targets using the classic sandwich mode and tagged with the SA-QDs, then the QDs-tagged beads floating in the suspension were directly trapped and excited by the optical tweezers to give strong and stable fluorescence signal, which was applied to quantify the targets. The distinctive size information from the image of the trapped beads enabled the sorting of the different targets. The results show that tiny laser power 40 μW is applicable for both trapping and fluorescence excitation of the beads. Moreover, the limits of detection for hemagglutinin7 (H7) gene and neuraminidase 9 (N9) gene are 1.0 – 2.0 pM with good selectivity for the complex sample, which is two orders of magnitude lower than that of the conventional method. More importantly, this strategy was successfully used to identify the subtype of the avian influenza A virus by simultaneous detection of H7 and N9 gene sequences. The high sensitivity, good selectivity, typing ability and the low cost of the laser make this strategy a promising method for life science and clinical applications.

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Simultaneous detection of biomolecular interactions and surface topography using photonic force microscopy

Seungjin Heo, Kipom Kim, Rodriguez Christophe, Tae-Young Yoon, Yong-Hoon Cho

We developed a photonic force microscope that can map multiple parameters simultaneously, including the surface topography and biomolecular interactions. To track the position of the probe bead and to determine contact position with the sample surface, we adopted a video analysis method using the diffraction pattern of monochromatic light passing through the probe bead. To demonstrate the capability of the microscope, we report the simultaneous measurement of the molecule distribution of DNA oligonucleotides on the surface, the binding strength of DNA hybridization between the bead and surface, and the topography of the smooth moulded surface.

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