Integrated Sample-to-Detection Chip for Nucleic Acid Test Assays
R. Prakasha, K. Pabbarajua, S. Wonga, R. Telliera,b*, K.V.I.S. Kalerc
a
Provincial Laboratory for Public Health of Alberta, Calgary, 3030 Hospital Drive NW, Calgary, AB T2N 4W4, Canada.
Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW Calgary, AB T2N 4N1,
Canada.
c
Department of Electrical and Computer Engineering, University of Calgary, 2500 University Drive NW Calgary, AB T2N1N4, Canada.
*Corresponding author: e-mail: raymond.tellier@albertahealthservices.ca
b
Characteristics of micro-beads and extraction/wash buffers:
Apart from the stock concentration and solid fraction measurements (), all other structural properties of the mag-bind micro-beads
were provided by the manufacturer. Estimation of the micro-bead concentration was required for the numerical analysis of the DEP
based micro-bead capture. The concentration was measured by loading a 1000X dilution of the stock solution in a Neubauer chamber
hemocytometer for counting, with the dilution performed using RNAse free water, in three steps of serial 5-fold dilutions. The
measured concentration and other characteristics of the mag-bind micro-beads are: concentration: 7500-8000 beads/L; Bead
diameter: ~ 2.0 m; Solid fraction (% by vol.): 1.15-1.25%; thickness of silica shell: ~ 50 nm.
The measured pH and conductivity values for the used buffer media are: 1. TNA Lysis Buffer:pH - 5.95, cond. - 156.2 mS/cm; 2.
VHB Wash Buffer:pH - 4.89,cond. - 42.5 mS/cm; 3. SPR Wash Buffer: pH - 7.98, cond. - 35.0 S/cm and RNAse Free Water (elution
media):pH - 6.38,cond. - 23.5 S/cm.
These micro-beads properties, along with the measured electrical and physical properties of the three buffers used in the lysis,
washing and elution steps were used in Eqns. 5 and 6 during the numerical analysis of the negative-DEP forces on the micro-beads;
they were also used in Eqns. 2, 3 and 4 for the estimation of the negative-DEP forces for DEP capture of the micro-beads, as reported
in the results and discussion section.
Analyzing the RT-PCR efficiency from the PMT recorded real-time PCR curve:
The efficiency of the different RT-PCR amplification assays were calculated for the clinical samples extracted on-chip and on the
easyMAG™ extractor at ProvLab Calgary. During the RT-PCR amplification process, fluorescence emission from the PCR droplet
increases with the amount of the amplified product. In this study, the normalized fluorescent emission values were captured in r ealtime using the ABI 7500 Fast RT-PCR equipment which was used for all the validation experiments during the course of this work.
The extracted and normalized fluorescence value was plotted with respect to cycling number to reproduce the standard PCR curves
from the equipment. The expected standard curve can be derived from Eqn. A.1.
A logarithmic plot of the RT-PCR curve yields a better visualization of the distinct reaction kinetics during the amplification process.
Ct (threshold cycle) is defined as the intersection between an amplification curve and a threshold line, placed in the qRT-PCR curves
above the signal noise floor. It can be shown to be related to the initial target concentration in the PCR reaction. Eqn. 1a describes the
exponential amplification of PCR products:
N n  Ni 1  E 
n
(A.1)
Where, Ni = initial copy number; N n = copy number at cycle n; n = number of cycles and E = efficiency of target amplification, with
theoretical values between 0 and 1. When the reaction efficiency is a maximum (E = 1 or 100%), the equation reduces to: N n = Ni*(2n)
and the target DNA copy count increase by 2-fold at each cycle. The quantity of PCR product generated at each cycle decreases with
decreasing efficiency, and the amplification plot is delayed. The measured efficiency (%) for successful and reliable PCR
amplification should lie between 90 and 110%.
In order to calculate the efficiency E of an RT-PCR assay, an interval with two end cycle numbers (n 1 and n2) were chosen from the
exponential region for which the efficiency (E) is defined. Eqn. A.1 is re-written using fluorescence output at the two PCR cycles as
seen in Eqn. A.2 and further re-arranged in Eqn. A.3, leading to a numerical expression for the efficiency, based entirely on the
analysis of the exponential region of the RT-PCR curve.
N n2
(n  n )
 1  E  2 1 ;
N n1
N 
1
log(1  E) 
Log  n2 
(n 2  n1 )
 N n1 


1
(A.2)


 N  ( n2  n1 
E   n2 
1
 N n1 
(A.3)
This method when applied to the experimental RT-PCR curves yielded PCR efficiencies in the range of (94.5 – 95.7 %) for chip
extracted samples and (94.7 – 96.8 %) for easyMag™ extracted samples. The comparable and acceptable values from the analyzed
clinical panel samples further establishes that the overall quality (therefore the absence of PCR inhibiting agents) of the chip extracted
nucleic acid samples is as good as the commercial extraction equipment.
The slope (m) of the linear, relative quantification curve (shown in Fig. 5b) is also related to the efficiency (E) of the PCR reaction as:
E  10 1/ m  1
(A.4)
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Based on the measured slope from Fig. 8b, the calculated RT-PCR efficiency for the chip based extraction of the four serial dilution
MS2 bacteriophage samples is ~ 96.4%.
MS2 validation experiments and outcomes:
Prior to using the sample preparation chip for nucleic acid extraction from patient samples, validation experiments were carr ied out to
establish and optimize the different assay parameters including lysis time, washing time, micro-bead capture and recollection time
during the sample preparation assay. Three different batches of laboratory prepared aliquots of MS2 bacteriophages were used in the
validation experiments. MS2 has RNA as its genomic nucleic acid, which is more labile and prone to degradation compared to DNA,
and thus a more stringent control for the extraction (Dreier et. al. 2005). Ten-fold serial dilutions of the MS2 phage (batch 1) spanning
over four logs of template concentration were extracted to mimic a wide range of viral loads. The four serial dilutions of MS2
(labelled as: 10-3 to 10-6) were extracted using the chip based nucleic acid sample preparation assay. The MS2 extracts were amplified
using the RT-PCR chip, previously reported in (Prakash et. al. 2015).
TableA.1. Volumetric and procedural details for the chip sample preparation in comparison to the benchmark extraction protocol, recommended for the Omega
bio-tek kit. (All reported volumes are in L).
Reagents
(Omega bio-tek)
Total Volume
(Mag-Bind® Viral DNA/RNA -200 uL
protocol)
Total Volume (chip protocol)
Lysis mastermix
TNA Lysis Buffer
Carrier RNA
Isopropanol
MS2 (Int. cont.)
240
8
280
10
24
1
27
1
Bead Mixture
Micro-bead
Proteinase K
10
10
1
1
Buffers and process
steps
Sample/Reagent
Total Volume
(Mag-Bind® Viral DNA/RNA -200 uL
protocol)
Total Volume (chip protocol)
Lysis mastermix
Bead Mix.
Sample
Total volume:
VHB Wash step
SPR Wash step
SPR Wash step
Elution step
538
20
200
758
400
500
500
110
53
2
20
75
20
20
20
12.5
1 (off-chip mixing)
2
3
4
5
Optimization of the chip based RNA extraction using MS2 Bacteriophage
In our theoretical analysis, we established that judicious selection of electro-actuation parameters (AC actuation voltage and
frequency) allows for the maneuvering of nucleic acid binding micro-beads using the negative-DEP force, assisted by the gravitational
sedimentation of the micro-bead within the droplet. However, along with the DEP capture parameters, the washing and elution times
are also critical aspects of a nucleic acid extraction system for effective removal of proteins, chaotropic salts, cellul ar debris and other
molecules which may have an inhibitory effect on the PCR amplification tests downstream (Dreier et. al., 2005). We used the MS2
Bacteriophage samples to optimize these steps for the chip based sample preparation assay.
Serial dilutions of the MS2 samples were used to optimize the key process parameters to ensure that comparable extraction
performance was achieved on chip. We first established that up to 30 mins of sample lysis time is sufficient. In all the experimental
studies reported in this work, samples were premixed with the lysis solution for transportation; the micro-bead mixture was added
within 30 minutes of the chip sample preparation experiments.
The washing and capture time was set for around 2 mins based on the analysis of Ct values corresponding to different washing/capture
times and was kept constant (2 mins) in all experiments. The elution time was increased up to 5 minutes based on experimental
observation to minimize the presence of micro-beads in the eluted sample which can possibly inhibit subsequent RT-PCR assays.
Fig. A.1 and Fig. A.2 show the RT-PCR results obtained using the chip extraction of the different panel samples and serial dilutions of
the MS2 suspension. Fig. A.1 cumulatively reports the measured Ct values from all the chip nucleic acid extraction experiments. The
RT-PCR curves for serial dilutions of MS2 samples are reported in Fig. 8a and the corresponding Ct values are plotted vs the
Normalized log10(MS2 conc.) in Fig. A.2(b).
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Fig. A.1: Scatter plot of the Ct values of all the internal control samples, included in extraction and RT-PCR experiments. Standard
deviation () values are numerically extracted for each of the batches of internal control, MS2 used during the chip experiments.
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Fig. A.2: (a) On-Chip RT-PCR curves for the serial dilution of stock MS2 sample; (b) Standard quantification curve for the optimized
chip based extraction of MS2 solutions; (c) concentration measurements (Nanodrop, ND-1000) for the amplified MS2 solutions
following chip based extraction and RT-PCR amplification assays.
As discussed previously, the standard quantification curve yielded a RT-PCR efficiency of ~96.4% for the MS2 extraction
experiments. The amplified products from each serial dilution were then measured with an absorption spectrophotometer (ND-1000,
Nanodrop, USA) and the normalized nucleic acid concentration in the amplified product (after 35 PCR cycles) plotted vs. the M S2
sample concentration (Fig. A.2(c)). The results of the chip based MS2 extractions, followed by the RT-PCR amplification assays
clearly demonstrate that the sensitivity achieved with the chip based extraction procedure is comparable to that obtained wit h the
easyMAG™ extraction platform.
References (Appendix 1):
J. Dreier, M. Stormer, K. Kleesiek, J. Clin. Microbio., 43, 4551-4557 (2005).
R. Prakash, K. Pabbaraju, S. Wong, A. Wong, R. Tellier, K.V.I.S. Kaler, Micromachines, 6, 63-79 (2015).
Online Resource: Caption for video files
Video 1: Illustrates model parameters used in the COMSOL multiphysics simulation of the negative-DEP capture of micro-beads in
an extraction buffer media and reports the static and time-dependant solver outputs (Electric potential, electric field plots and particle
tracing during negative-DEP capture). Vact: Actuation voltage (Vpp); fact: Actuation frequency (Hz).
Video 2: Snapshots of the chip based, four-plex nucleic acid extraction scheme; actuation videos showing four steps of the on-chip
nucleic acid extraction assay; Step 1: Droplet electro-actuation and transport of micro-beads (with bound nucleic acid) in extraction
buffer (VHB wash buffers); Step 2: Negative-DEP capture of micro-beads from VHB wash buffer; Step 3: removal of waste solution
following micro-bead capture; Step 4: Washing and re-collection of micro-beads into SPR wash buffer.
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