Lab on a single microbead: an ultrasensitive detection strategy enabling microRNA analysis at the single-molecule level† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc02641e

A single microbead-based sensing platform has been developed, which enables the detection of microRNA at the single-molecule level.


Reagents and Materials
Biotinylated EXPAR templates, all of the target miRNAs, mixture of dNTPs, RNase inhibitor and RNase-free water were purchased from TaKaRa (Dalian, China). The Vent (exo-) polymerase, Nt.BtsNBI nicking enzyme, and the ThermoPol buffer were obtained from New England BioLabs

Standard Protocols of the EXPAR-SMBS Platform for Let-7a Analysis
Conjugation of the EXPAR templates on the microbeads. After thoroughly blending, 10 μL slurry of the Streptavidin Mag Sepharose microbeads (STV-beads) was pipetted into a 1.5 mL centrifuge tube and washed twice with TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) via magnetic isolation.
Afterward, an excess amount of biotinylated EXPAR template (1 nmol) was incubated with the STVbeads for 30 min under shaking. Then these microbeads were magnetically separated to remove the S-3 excess unbound EXPAR template and washed three times with RNase-free water. Finally, the template-conjugated microbeads were re-suspended in 500 μL of RNase-free water and ready for subsequent use.  Fig. S1 illustrates the catching process of a single microbead.

Manipulation of a single microbead.
A hydrophobic 96-well plate cover with transparent property can keep about 100 μL water (containing 2 μL of EXPAR template-conjugated beads) in the shallow wells (image A), which is an ideal platform to assist the capture of individual microbeads. A Narishige micromanipulator system equipped on an Olympus IX53 inverted microscope with a monitor is used to manipulate a single microbead (image B~F). With the help of the camera and monitor, one can search the suitable microbeads on the screen by naked eyes (image B). A DIY ruler (image C) is pasted on the screen to help us select the beads with the desired narrow size (80 ± 5 μM). Once the desirable microbead is selected from the screen, we only need a pipette to catch it (in 1 μL volume, image D~F) and the manipulation of a single microbead only needs 1~2 min.

S-4
Standard EXPAR-SMBS procedures. The reaction mixtures for the EXPAR-SMBS system were prepared separately on ice as part A and part B. Part A consisted of 0.5× Nt.BstNBI buffer, 0.5× ThermoPol buffer, RNase inhibitor and the miRNA target. Part B consisted of 0.5× ThermoPol buffer, dNTPs, biotin-dATP, Nt.BstNBI nicking endonuclease, Vent (exo -) DNA polymerase, and RNase-free water.
Moreover, for the single-cell let-7a analysis, a single HCT-116 cell was picked up by a capillary (diameter of 100 μm) with the micromanipulator system (Narishige, equipped on an Olympus IX53 microscope). Then the single cell was suspended into 1 μL of RNase-free water and lysed at 95ºC for 5 min, which was immediately used as the sample to perform the EXPAR-SMBS reaction according to S-5 the standard procedures stated above.
Fluorescence imaging test. All images were taken using an Olympus FV-1200 laser scanning confocal microscope. The microbead in 5 μL of PBS buffer was spread on a cover slip and the fluorescence image was obtained by collecting the fluorescence at the wavelength range from 565 nm to 665 nm under the excitation of a 559 nm laser. In particular, in order to acquire the quantitative value of a fluorescent microbead for quantitative analysis of miRNAs, we made a z-stack scan to cut the depth of a single microbead into 10 slices (see illustration in Fig. S2), and the integrated fluorescence intensities of these slices were counted together to avoid the possible errors during manual focusing. It also should be noted that for the quantitative analysis of miRNAs, the maximum fluorescence value of a bright spot that can be quantitatively acquired by the fluorescence microscope is 4096. In order to acquire brighter microbead image but not exceed this maximum value, the PMT HV of the fluorescence microscope may be reasonably tuned for the imaging of microbeads at different batches, and the used PMT HV values are provided in the corresponding figure captions in the manuscript.

Optimization of the Amount of Vent (exo-) DNA Polymerase and the Nicking Enzyme
According to the principle of EXPAR, the amplification efficiency will be seriously influenced by the amount of DNA polymerase and nicking enzyme. The effect of the amount of Vent (exo -) polymerase was first investigated in this study. The integrated fluorescence signal of the microbead treated by 10 fM let-7a was recorded respectively by using 0.01 U/μL, 0.015 U/μL, 0.02 U/μL, 0.03 U/μL Vent (exo -) DNA polymerase. The blank was treated under the same procedures without let-7a. It can be seen from Fig. S3 that with the increase of Vent (exo -) polymerase, the reaction time, at which the fluorescence signals produced by 10 fM let-7a can be clearly discriminated from the blank, is gradually shortened. In consideration of all of these parameters such as high microbead brightness, high S/B ratio and short assay time, 0.02 U/μL Vent (exo -) DNA polymerase with a 42 min reaction time was selected to be the optimum.  Error bars represents the standard deviation from three repetitive measurements.