TY - JOUR
T1 - A modular and reconfigurable open-channel gated device for the electrokinetic extraction of cell-free DNA assays
AU - Futai, Nobuyuki
AU - Fukazawa, Yuto
AU - Kashiwagi, Tomohiro
AU - Tamaki, Shogo
AU - Sakai, Riho
AU - Hogan, Catherine A.
AU - Murugesan, Kanagavel
AU - Ramachandran, Ashwin
AU - Banaei, Niaz
AU - Santiago, Juan G.
N1 - Funding Information:
As shown in Fig. 1, the four gel gates from the negative electrode to the positive were labeled as G0, G1, G2, and G3. Gates G0, G1, and G2, were each formed with 26 μl of 1% agarose (PrimeGel Agarose LE 1–20K, Takara Bio) dissolved in a buffer solution. The G3 gate was fabricated with the same procedure, except 3% of a low-melting temperature (LMT) high-resolution agarose called PCR agarose (PrimeGel Agarose LMT PCR-Sieve GAT, Takara Bio) was used instead of standard agarose. The buffer solutions for G0, G1, G2, and G3 were TE, LE (adjusted to pH 7.5), LE (pH 8.0), and LE (pH 8.0), respectively. Four types of agarose solution were stored in 5-ml conical tubes and heated in 90 °C water. They were then dispensed into separate gate mold slots in the PDMS slab and allowed to cool to room temperature to form solidified agarose (see the details in SI.1 in the Supporting Information).The position of the ITP zone was tracked using a dark band of Ponceau S. Immediately after the Ponceau-S fully exited from the G3 gate and no red color of Ponceau S was visible within gate G3, the voltage was manually deactivated. Then gate G3 was immediately retracted and then deposited into a PCR tube (already containing the master mix described in Section 2.6) using a pair of tweezers (SM103, Ideal-tek; see SI.2 in the Supporting Information).In the present study, it is hypothesized that the effects of the proteinase K concentration on the plasma samples result from the creation of a fraction of a high-mobility peptide species. The increase in spacing between the Ponceau-S band and the DNA may be due to proteinase K products acting as endogenous ITP spacer molecules that focus on the Ponceau S and the 100-bp DNA in the ITP stage of the process. Note that proteinase K itself has a pI of 8.9, while the pH of our trailing electrolyte was about pH8.3, according to a simulation result from CAFES web-based ITP simulator [40,41]. Hence, we do not expect Proteinase K to focus in anionic ITP. Consistent with this hypothesis is an observable gap between these peaks at this stage of the experiment. Evidence of the latter spacing (in the ITP region of the chip) is shown and discussed in the Supporting Information (SI.4). Note that most plasma proteins have relatively low pI values [42] and are expected to migrate in the same direction as the DNA in basic (>pH 8) buffers. This suggests that a fraction of small peptides [43,44] created from these proteins have sufficiently high anionic mobility to act as ITP electrophoretic spacers (See SI.5).In all cases, the tITP approach was able to separate and resolve the 100- and 200-bp DNA from the Ponceau-S marker in the plasma samples. However, as discussed in the Supporting Information (SI.6), in the experiments with plasma but without proteinase K treatment, there was a considerable amount of DNA that could not be recovered. Therefore, a concentration of proteinase K of 1500 μg/ml and a plasma volume of 10 μl were chosen for the MTB assay described in the next section.This work was supported by JSPS KAKENHI Grant Number 16K01374. JGS, NB, and AR also gratefully acknowledge funding from Stanford's Interdisciplinary Biosciences Institute (Bio-X).
Funding Information:
JGS, NB, and AR also gratefully acknowledge funding from Stanford's Interdisciplinary Biosciences Institute (Bio-X).
Funding Information:
This work was supported by JSPS KAKENHI Grant Number 16K01374.
Publisher Copyright:
© 2022 Elsevier B.V.
PY - 2022/4/1
Y1 - 2022/4/1
N2 - The high-efficiency separation and extraction of short fragments of cell-free DNA (cfDNA) remain challenging due to their low abundance and short lengths. This study presents a method for separating short cfDNA fragments, with lengths ranging from about 100 to 200 base pairs, from liquid human plasma samples into separable and extractable bands as solid agarose gel slabs. To achieve this, a novel millimeter-scale fluidic device is used for sample handling, transient isotachophoresis, and extraction. The device features open-to-atmosphere liquid chambers that define and manually actuated (i.e., movable) agarose-made gate valve structures. The agarose gates then define discrete zones for buffers, sample injection, DNA pre-concentration via isotachophoresis, size-based gel separation, and DNA-band extraction. As a demonstration of its efficacy, the device is applied to the enrichment and purification of M. tuberculosis genomic DNA fragments spiked in human plasma samples. This purified cfDNA is analyzed using the quantitative polymerase chain reaction (qPCR) of the IS6110 repetitive sequence in the M. tuberculosis genome. The data from this study demonstrates that high sensitivity can be achieved in cfDNA detection, as shown by the comparison with a typical solid-phase extraction method and buffer spiked with cfDNA. Evidence is presented that suggests plasma peptides generated by treatment of the sample with proteinase K acts as endogenous spacer molecules, which improve the resolution and purification of DNA relative to the marker dye and other contaminants that decrease the signal level in qPCR.
AB - The high-efficiency separation and extraction of short fragments of cell-free DNA (cfDNA) remain challenging due to their low abundance and short lengths. This study presents a method for separating short cfDNA fragments, with lengths ranging from about 100 to 200 base pairs, from liquid human plasma samples into separable and extractable bands as solid agarose gel slabs. To achieve this, a novel millimeter-scale fluidic device is used for sample handling, transient isotachophoresis, and extraction. The device features open-to-atmosphere liquid chambers that define and manually actuated (i.e., movable) agarose-made gate valve structures. The agarose gates then define discrete zones for buffers, sample injection, DNA pre-concentration via isotachophoresis, size-based gel separation, and DNA-band extraction. As a demonstration of its efficacy, the device is applied to the enrichment and purification of M. tuberculosis genomic DNA fragments spiked in human plasma samples. This purified cfDNA is analyzed using the quantitative polymerase chain reaction (qPCR) of the IS6110 repetitive sequence in the M. tuberculosis genome. The data from this study demonstrates that high sensitivity can be achieved in cfDNA detection, as shown by the comparison with a typical solid-phase extraction method and buffer spiked with cfDNA. Evidence is presented that suggests plasma peptides generated by treatment of the sample with proteinase K acts as endogenous spacer molecules, which improve the resolution and purification of DNA relative to the marker dye and other contaminants that decrease the signal level in qPCR.
KW - Agarose gel electrophoresis
KW - Cell-free DNA
KW - DNA purification
KW - isotachophoresis
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U2 - 10.1016/j.aca.2022.339435
DO - 10.1016/j.aca.2022.339435
M3 - Article
C2 - 35256135
AN - SCOPUS:85125138162
SN - 0003-2670
VL - 1200
JO - Analytica Chimica Acta
JF - Analytica Chimica Acta
M1 - 339435
ER -