Asynchronous Magnetic Bead Rotation (AMBR) Microviscometer for Label-Free DNA Analysis

Abstract

We have developed a label-free viscosity-based DNA detection system, using paramagnetic beads as an asynchronous magnetic bead rotation (AMBR) microviscometer. We have demonstrated experimentally that the bead rotation period is linearly proportional to the viscosity of a DNA solution surrounding the paramagnetic bead, as expected theoretically. Simple optical measurement of asynchronous microbead motion determines solution viscosity precisely in microscale volumes, thus allowing an estimate of DNA concentration or average fragment length. The response of the AMBR microviscometer yields reproducible measurement of DNA solutions, enzymatic digestion reactions, and PCR systems at template concentrations across a 5000-fold range. The results demonstrate the feasibility of viscosity-based DNA detection using AMBR in microscale aqueous volumes.

Keywords: DNA; genetic analysis; label-free; magnetic; microdevice; paramagnetic; quantitative PCR; restriction digestion; viscometer; viscosity.

Figure 1

Asynchronous magnetic bead rotation (AMBR) microviscometer. (a) A schematic experimental set-up of an AMBR microviscometer. 1: perpendicular Helmholtz coils for rotating field generation; 2: liquid to be measured; 3: magnetic bead; 4: inverted microscope objective. (b) Observed bead rotation frequency vs. field driving frequency. Below 9 Hz the bead rotation frequency matches that of the field; above 9 Hz, the bead rotates asynchronously, with frequency decreasing as the driving frequency increases. (c) Viscosity measurement of glycerol/water mixture solutions. The graph compares AMBR results in a magnetic field with 100 Hz driving frequency to published values and conventional (Ubbelohde) viscometer measurements of the same liquid. (d) AMBR microviscometer linear response to viscosity in prepared solutions of glycerol/water at 100 Hz driving frequency. Error bars represents standard deviation among three measurements.

Figure 2

Reproducibility of AMBR viscosity measurements at 100 Hz driving frequency. (a) Rotation period measurement of 20 independent beads in the same solution plotted against the optically measured bead size of each bead. (b) The rotation periods of two examples of 45 µm beads observed over time in the same solution. The rotation periods are calculated over a 12 s period and plotted in the graph. The average values are for 17 sequential observations

Figure 3

DNA measurement using AMBR microviscometer. (a) Viscosities of lambda DNA EcoRI digest solutions at different concentrations, as measured by AMBR microviscometer. The green area indicates the expected range of the viscosity calculated theoretically, assuming that only the longest (top range) or only the shortest (bottom range) DNA fragment size is present. Error bars represent standard deviation among 10 beads in one measurement. (b) Measurement of bead rotation period of pre- and post-digestion samples of lambda DNA by AMBR microviscometer. The field driving frequency is 150 Hz. The error bars show the standard deviation among 10 beads in each measurement. (c) Measurement of viscosity by bead rotation period in PCR reactions sampled every 5 cycles, starting from the 6th cycle. PCR reactions with initial DNA amounts of 0 ng, 0.05 ng, 5 ng, 55 ng, and 250 ng are shown. The reaction volumes are 50 µL each. The field driving frequency is 150 Hz, and the PCR product size is 4500 bp. Each point represents the mean value, observing ten beads. (d) Fluorescent signal intensities of the PCR product (4500 bp band) observed on a electrophoresis gel for the same samples measured in (c).

Figure A1

Images of the bead rotation at time 0, ¹/₄ T, ¹/₄ T, ³/₄ T and T for 45 µm beads at 100 Hz driving frequency, where T is the bead rotation period. While the commercial beads look spherical and symmetrical by eye, the software can tell the subtle difference in shape and surface smoothness of the beads, so as to determine the bead rotation periods.

Figure A2

Calibration curves of 45µm bead at different driving frequencies. A: 30 Hz; B: 100 Hz; C: 200 Hz; D: 250 Hz. The critical frequency is at 10–15 Hz. The error bars represent the standard deviation among ten different beads in one measurement. The calibration curves yield good linearity consistently at frequencies away from the critical frequency, i.e., from 100 Hz to 250 Hz.

Figure A3

Calibration curves at 100 Hz using beads of different sizes. A: 7.6 µm; B: 16 µm; C: 45 µm. Error bars represents standard deviation among 10 beads in one measurement.

Figure A4

Plot of reaction cycle number versus log of initial DNA amount for the qPCR measurement by AMBR method. Error bars represent the uncertainty due to the AMBR measurement of every five cycles.