Limits...
Electrical detection of nucleic acid amplification using an on-chip quasi-reference electrode and a PVC REFET.

Salm E, Zhong Y, Reddy B, Duarte-Guevara C, Swaminathan V, Liu YS, Bashir R - Anal. Chem. (2014)

Bottom Line: Here we demonstrate a novel method of utilizing a microfabricated solid-state quasi-reference electrode (QRE) paired with a pH-insensitive reference field effect transistor (REFET) for detection of real-time pH changes.The end result is a 0.18 μm, silicon-on-insulator, foundry-fabricated sensor that utilizes a platinum QRE to establish a pH-sensitive fluid gate potential and a PVC membrane REFET to enable pH detection of loop mediated isothermal amplification (LAMP).This technique is highly amendable to commercial scale-up, reduces the packaging and fabrication requirements for ISFET pH detection, and enables massively parallel droplet interrogation for applications, such as monitoring reaction progression in digital PCR.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.

ABSTRACT
Electrical detection of nucleic acid amplification through pH changes associated with nucleotide addition enables miniaturization, greater portability of testing apparatus, and reduced costs. However, current ion-sensitive field effect transistor methods for sensing nucleic acid amplification rely on establishing the fluid gate potential with a bulky, difficult to microfabricate reference electrode that limits the potential for massively parallel reaction detection. Here we demonstrate a novel method of utilizing a microfabricated solid-state quasi-reference electrode (QRE) paired with a pH-insensitive reference field effect transistor (REFET) for detection of real-time pH changes. The end result is a 0.18 μm, silicon-on-insulator, foundry-fabricated sensor that utilizes a platinum QRE to establish a pH-sensitive fluid gate potential and a PVC membrane REFET to enable pH detection of loop mediated isothermal amplification (LAMP). This technique is highly amendable to commercial scale-up, reduces the packaging and fabrication requirements for ISFET pH detection, and enables massively parallel droplet interrogation for applications, such as monitoring reaction progression in digital PCR.

Show MeSH

Related in: MedlinePlus

End point detection of LAMP on-chip. (a) I–V curves of a PVC-REFET deviceare shown. The differencesbetween the negative control before and after amplification are notsignificant. The positive control shows a shift to a higher thresholdvoltage, which is consistent with a decrease in pH. The pH changewas measured to be −1.2 units with a commercial meter. (b)Measurements were taken simultaneously with a non-PVC ISFET. The positiveand negative amplification solutions show insignificant differences.(c) The change in threshold voltage was quantified for multiple devices.The change for positive amplification was statistically significant(n = 3, p-value < 0.01) whencompared to the negative control for the PVC-REFET. The non-PVC ISFETshowed no significant changes.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215847&req=5

fig5: End point detection of LAMP on-chip. (a) I–V curves of a PVC-REFET deviceare shown. The differencesbetween the negative control before and after amplification are notsignificant. The positive control shows a shift to a higher thresholdvoltage, which is consistent with a decrease in pH. The pH changewas measured to be −1.2 units with a commercial meter. (b)Measurements were taken simultaneously with a non-PVC ISFET. The positiveand negative amplification solutions show insignificant differences.(c) The change in threshold voltage was quantified for multiple devices.The change for positive amplification was statistically significant(n = 3, p-value < 0.01) whencompared to the negative control for the PVC-REFET. The non-PVC ISFETshowed no significant changes.

Mentions: To demonstrate this potential, we have shown detection ofa LAMPreaction using end-point pH measurements with a solid-state electrodeand an ISFET passivated with PVC. Macroscale pH measurements withthe In-Lab Ultra Micro pH meter showed a pH change of −1.24units for the complete full reaction. As shown in Figure 5b and c, without the PVC membrane, the positiveand negative amplification reactions are indistinguishable (two-tailed p-value = 0.7745). Compared to the negative reaction, thePVC REFET shows strong results that are statistically significant(p-value < 0.01). The PVC-REFET responds to thechange in the electrode potential, which is sensitive to the pH ofthe solution. Supporting Information FigureS-7 shows the stability of the device threshold voltage with repeatedsolution exchanges. The LAMP end-point data was statistically significantcompared to variations in the threshold voltage associated with solutionexchange (p-value < 0.0001).


Electrical detection of nucleic acid amplification using an on-chip quasi-reference electrode and a PVC REFET.

Salm E, Zhong Y, Reddy B, Duarte-Guevara C, Swaminathan V, Liu YS, Bashir R - Anal. Chem. (2014)

End point detection of LAMP on-chip. (a) I–V curves of a PVC-REFET deviceare shown. The differencesbetween the negative control before and after amplification are notsignificant. The positive control shows a shift to a higher thresholdvoltage, which is consistent with a decrease in pH. The pH changewas measured to be −1.2 units with a commercial meter. (b)Measurements were taken simultaneously with a non-PVC ISFET. The positiveand negative amplification solutions show insignificant differences.(c) The change in threshold voltage was quantified for multiple devices.The change for positive amplification was statistically significant(n = 3, p-value < 0.01) whencompared to the negative control for the PVC-REFET. The non-PVC ISFETshowed no significant changes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215847&req=5

fig5: End point detection of LAMP on-chip. (a) I–V curves of a PVC-REFET deviceare shown. The differencesbetween the negative control before and after amplification are notsignificant. The positive control shows a shift to a higher thresholdvoltage, which is consistent with a decrease in pH. The pH changewas measured to be −1.2 units with a commercial meter. (b)Measurements were taken simultaneously with a non-PVC ISFET. The positiveand negative amplification solutions show insignificant differences.(c) The change in threshold voltage was quantified for multiple devices.The change for positive amplification was statistically significant(n = 3, p-value < 0.01) whencompared to the negative control for the PVC-REFET. The non-PVC ISFETshowed no significant changes.
Mentions: To demonstrate this potential, we have shown detection ofa LAMPreaction using end-point pH measurements with a solid-state electrodeand an ISFET passivated with PVC. Macroscale pH measurements withthe In-Lab Ultra Micro pH meter showed a pH change of −1.24units for the complete full reaction. As shown in Figure 5b and c, without the PVC membrane, the positiveand negative amplification reactions are indistinguishable (two-tailed p-value = 0.7745). Compared to the negative reaction, thePVC REFET shows strong results that are statistically significant(p-value < 0.01). The PVC-REFET responds to thechange in the electrode potential, which is sensitive to the pH ofthe solution. Supporting Information FigureS-7 shows the stability of the device threshold voltage with repeatedsolution exchanges. The LAMP end-point data was statistically significantcompared to variations in the threshold voltage associated with solutionexchange (p-value < 0.0001).

Bottom Line: Here we demonstrate a novel method of utilizing a microfabricated solid-state quasi-reference electrode (QRE) paired with a pH-insensitive reference field effect transistor (REFET) for detection of real-time pH changes.The end result is a 0.18 μm, silicon-on-insulator, foundry-fabricated sensor that utilizes a platinum QRE to establish a pH-sensitive fluid gate potential and a PVC membrane REFET to enable pH detection of loop mediated isothermal amplification (LAMP).This technique is highly amendable to commercial scale-up, reduces the packaging and fabrication requirements for ISFET pH detection, and enables massively parallel droplet interrogation for applications, such as monitoring reaction progression in digital PCR.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.

ABSTRACT
Electrical detection of nucleic acid amplification through pH changes associated with nucleotide addition enables miniaturization, greater portability of testing apparatus, and reduced costs. However, current ion-sensitive field effect transistor methods for sensing nucleic acid amplification rely on establishing the fluid gate potential with a bulky, difficult to microfabricate reference electrode that limits the potential for massively parallel reaction detection. Here we demonstrate a novel method of utilizing a microfabricated solid-state quasi-reference electrode (QRE) paired with a pH-insensitive reference field effect transistor (REFET) for detection of real-time pH changes. The end result is a 0.18 μm, silicon-on-insulator, foundry-fabricated sensor that utilizes a platinum QRE to establish a pH-sensitive fluid gate potential and a PVC membrane REFET to enable pH detection of loop mediated isothermal amplification (LAMP). This technique is highly amendable to commercial scale-up, reduces the packaging and fabrication requirements for ISFET pH detection, and enables massively parallel droplet interrogation for applications, such as monitoring reaction progression in digital PCR.

Show MeSH
Related in: MedlinePlus