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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.

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FET responsewith platinum fluid gate. (a) A real-time pH responsecurve of a PVC and non-PVC device with 4 subsequent HCl additionsis shown. The non-PVC ISFET shows almost zero response when operatedby the platinum electrode. (b) The pH response is quantified for manydevices across multiple chips (n = 3). The PVC REFETsensor shows an apparent opposite trend than expected with hydrogenaddition. This is due to the charge at the platinum electrode beingmirrored into the REFET response. (c) A real-time pH response curvewith four subsequent NaOH additions is shown. (d) The pH responseis quantified across multiple devices (n = 3). Thenon-PVC ISFET shows minimal pH response compared to the PVC-REFET.
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fig2: FET responsewith platinum fluid gate. (a) A real-time pH responsecurve of a PVC and non-PVC device with 4 subsequent HCl additionsis shown. The non-PVC ISFET shows almost zero response when operatedby the platinum electrode. (b) The pH response is quantified for manydevices across multiple chips (n = 3). The PVC REFETsensor shows an apparent opposite trend than expected with hydrogenaddition. This is due to the charge at the platinum electrode beingmirrored into the REFET response. (c) A real-time pH response curvewith four subsequent NaOH additions is shown. (d) The pH responseis quantified across multiple devices (n = 3). Thenon-PVC ISFET shows minimal pH response compared to the PVC-REFET.

Mentions: Initial ID–VG curvesmeasuring drain current, while sweeping the fluid gate were extractedwith first the reference electrode and then with the quasi-referenceplatinum electrode. For pH titrations, current vs time measurementswere taken with the FET set in the linear regime. To standardize measurementsfor different devices, the measured current was compared to the baseline ID–VG curveto extract the extended gate surface potential (see Figure 1d and f and Figure 2).


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)

FET responsewith platinum fluid gate. (a) A real-time pH responsecurve of a PVC and non-PVC device with 4 subsequent HCl additionsis shown. The non-PVC ISFET shows almost zero response when operatedby the platinum electrode. (b) The pH response is quantified for manydevices across multiple chips (n = 3). The PVC REFETsensor shows an apparent opposite trend than expected with hydrogenaddition. This is due to the charge at the platinum electrode beingmirrored into the REFET response. (c) A real-time pH response curvewith four subsequent NaOH additions is shown. (d) The pH responseis quantified across multiple devices (n = 3). Thenon-PVC ISFET shows minimal pH response compared to the PVC-REFET.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: FET responsewith platinum fluid gate. (a) A real-time pH responsecurve of a PVC and non-PVC device with 4 subsequent HCl additionsis shown. The non-PVC ISFET shows almost zero response when operatedby the platinum electrode. (b) The pH response is quantified for manydevices across multiple chips (n = 3). The PVC REFETsensor shows an apparent opposite trend than expected with hydrogenaddition. This is due to the charge at the platinum electrode beingmirrored into the REFET response. (c) A real-time pH response curvewith four subsequent NaOH additions is shown. (d) The pH responseis quantified across multiple devices (n = 3). Thenon-PVC ISFET shows minimal pH response compared to the PVC-REFET.
Mentions: Initial ID–VG curvesmeasuring drain current, while sweeping the fluid gate were extractedwith first the reference electrode and then with the quasi-referenceplatinum electrode. For pH titrations, current vs time measurementswere taken with the FET set in the linear regime. To standardize measurementsfor different devices, the measured current was compared to the baseline ID–VG curveto extract the extended gate surface potential (see Figure 1d and f and Figure 2).

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