Performance Evaluation of Fast Microfluidic Thermal Lysis of Bacteria for Diagnostic Sample Preparation

Abstract

Development of new diagnostic platforms that incorporate lab-on-a-chip technologies for portable assays is driving the need for rapid, simple, low cost methods to prepare samples for downstream processing or detection. An important component of the sample preparation process is cell lysis. In this work, a simple microfluidic thermal lysis device is used to quickly release intracellular nucleic acids and proteins without the need for additional reagents or beads used in traditional chemical or mechanical methods (e.g., chaotropic salts or bead beating). On-chip lysis is demonstrated in a multi-turn serpentine microchannel with external temperature control via an attached resistive heater. Lysis was confirmed for Escherichia coli by fluorescent viability assay, release of ATP measured with bioluminescent assay, release of DNA measured by fluorometry and qPCR, as well as bacterial culture. Results comparable to standard lysis techniques were achievable at temperatures greater than 65 °C and heating durations between 1 and 60 s.

Keywords: bacterial detection; lab-on-a-chip; microfluidics; on-chip diagnostics; sample preparation; thermal lysis.

Figure 1

Silicon and glass microfluidic lysis chip—layout in Tanner L-edit software (top) and finished device (bottom). The top device has a 1 mm wide channel and the bottom device an 0.5 mm wide channel. The external chip dimensions are 70.5 mm × 9 mm × 1 mm. The rectangle marked “ROI” denotes the imaging area used for the BacLight live:dead membrane permeabilization assay (see Section 2.6).

Figure 2

Lysis performance as indicated by a decrease in colony forming units compared with a bead-beaten positive control (– – –). (a) For a range of on-chip residence times from 15 to 60 s at room temperature (25 °C) and at maximum heat (90 °C). (b) For the full temperature range for 15 s residence time each.

Figure 3

Membrane permeability as detected by the Baclight live/dead assay and measured as red:green ratios. (a) For a range of on-chip residence times from 15 to 60 s at room temperature (25 °C) and maximum heat (90 °C). (b) For the full temperature range for 15 s residence time each.

Figure 4

Percent extracellular ATP determined by chemiluminescent signal compared with a bead-beaten positive control (– – –). (a) For a range of on-chip residence times from 15 to 60 s at room temperature (25 °C) and maximum heat (90 °C). (b) For the full temperature range for 15 s residence time each.

Figure 5

Quantity of DNA recovered as reported by Qubit PicoGreen assay. (a) For a range of on-chip residence times from 15 to 60 s at room temperature (25 °C) and maximum heat (90 °C). (b) For the full temperature range for 15 s residence time each.

Figure 6

ΔC_T by qPCR compared to bead-beaten control (– – –). (a) For a range of on-chip residence times from 15 to 60 s at room temperature (25 °C) and maximum heat (90 °C). (b) For three temperatures, each with a 15 s residence time.

Figure 7

PCR-available DNA yield relative to an unprocessed sample (ΔC_T = 0), resulting from long off-chip sample heating times and short on-chip heating times, compared with bead-beating.

Figure 8

A qualitative scale of biophysical changes that occur in bacterial cells during lysis and the relative energy input required to attain them. At the top of the figure, each assay used in this work is linked to the approximate lysis step that it quantifies.