Fig 1.
Functional schematic of the RZTC wheel.
A fixed tube or capillary containing the sample to be cycled is held against temperature-controlled heater blocks, which are sequentially rotated into contact with the tube to produce the desired temperature cycling. Blocks are arranged around the wheel in order of increasing temperature. The tube rests in a groove on the outer surface of the heater block (right) and is tensioned against the block to maximize thermal coupling and sample ramp rate.
Fig 2.
Rotary zone thermal cycler prototype.
(A) Aluminum heater blocks with seven capillary grooves for parallel operation feature embedded cartridge heaters and RTDs interfaced to a printed circuit board and wire harness. Guide posts to either side of the heated zone have through-holes to maintain capillary alignment in the groove and provide options for tensioning the tube against the wheel. (B) Disassembled RZTC wheel showing insulating hub structure and heater block geometry. Axial holes in the heater block are for mounting screws (large inner pair), cartridge heaters (smaller outer pair), and RTD (smallest central hole).
Fig 3.
Functional fluidic schematic of the rzPCR system.
A single automated syringe pump and multiport valve provide for sample loading, positioning, and unloading as well as between-run cleaning of the sample inlet and reactor tube. Upstream and downstream 3-way valves lock the sample in position over the center of the wheel during cycling.
Fig 4.
Integrated rotary zone PCR system corresponding to the schematic of Fig. 3.
The wheel is oriented horizontally with the wire harness for heater power and temperature sensor measurements routed upward to the four-channel Omega controller box (Fig. 5B) on a shelf above the system. The sample capillary is tensioned against the upper part of the RZTC wheel by Sandia TubeTite connectors at the three-way valves upstream and downstream of the heated zone. Not visible in the photo are the Keyspan serial hub and RS485 converters behind the syringe pump.
Fig 5.
(A) Detail of the sample inlet and reagent distribution portion of the rzPCR system. Sample is aspirated into the capillary shown in the foreground, while the capillary in the back will deliver PCR reagents to a DMF platform in future iterations of the system. Reagent, flushing, and waste reservoirs plumbed to the central multiport valve can be readily emptied or refilled as needed. (B) Four-channel heater control box.
Fig 6.
rzPCR system communication and hardware control architecture.
A laptop-based DAQtrol graphical user interface (S3 Fig.) provides control and automation of the rzPCR system via pre-programmed scripts or manual control over pump, valve, heater, and data-logger operation.
Table 1.
Feature comparison of rzPCR cleaning protocols.
Fig 7.
Thermal cycling performance comparison for three thermal cycler designs between 60°C and 96°C.
Conventional Peltier based bench-top and hybrid Peltier/forced air OpenPCR thermal cyclers display significantly slower temperature ramp rates than those obtained by the stepper motor actuation of the RZTC.
Fig 8.
Comparison of average thermocouple-measured rise and fall times derived from the five cycles depicted in Fig. 7 with a parametrically fitted bounded exponential curve of the form of Equation 1, and response curves predicted analytically a priori from Equations 1, 2, and 4.
Table 2.
Measured and analytically predicted transitions between 60°C and 96°C for samples in polycarbonate (PC) and FEP tubing with and without a thermocouple (TC) present.
Fig 9.
RZTC power and temperature history from startup for six Phusion rzPCR experiments with thermal cycling intervals indicated by the shaded bands.
(A) Total (wall) power drawn by the heater control box during initial idle, warm-up, and cycling. (B) Detail of power fluctuations during cleaning, sample load, amplification, and sample unload. (C) Temperature fluctuations for heater blocks 2–4 about their set-points at 60°C, 72°C, and 98°C. (D) Temperature evolution of unheated block 1 over time.
Fig 10.
4-block RZTC temperature history from startup through a series of four PowerPlex 16 HS runs (shaded) with unloading, cleaning, and loading operations between them.
Block 3 is switched in real time between 96°C hot start and 90°C cycling set-points.
Fig 11.
E-Gel image showing alternating positive control and no template control 26-cycle GAPDH / Phusion experiments.
Lanes (M) 50 bp ladder, (1) Bench-top NTC, (2) Bench-top positive control, (3, 5, 7, 9) rzPCR NTC, (4, 6, 8, 10) rzPCR positive control. The yellow arrow indicates the position of primer bands, while green arrows indicate faint nonspecific bands in the rzPCR positive control lanes. Streaks and smudging are post-separation handling artifacts.
Fig 12.
E-Gel image showing sequential 26-cycle Phusion / GAPDH cleaning and decontamination experiments.
Odd numbered lanes are identical positive controls preceded by cleaning protocol A. Lanes (M) 50 bp ladder, (2) NTC preceded by cleaning protocol A, (4) NTC preceded by cleaning protocol B, (6) NTC preceded by cleaning protocol C, (8) NTC preceded by cleaning protocol D, (10) NTC preceded by cleaning protocol E. The yellow arrow indicates the position of primer bands, while green arrows indicate faint nonspecific bands in the rzPCR positive control lanes.
Fig 13.
E-Gel image showing PowerPlex 16 HS amplification results.
Lanes (M) 50 bp ladder, (1) Bench-top NTC, (2) Bench-top positive control 2800M template, (3) rzPCR positive control 2800M template, (4) rzPCR NTC.
Fig 14.
BioAnalyzer data from analysis of second-strand cDNA products generated by the bench-top and rzPCR systems.
Note that rzPCR reactions were run for an additional cycle (12 cycles total, as compared to 11 cycles for the bench-top system).