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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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Platinum surface potentialresponse. (a) A schematic of the opencircuit potential method is shown. A PDMS well is placed on a platinumsurface and Tris-HCl is added. The on-chip platinum (SMU1) vs thereference electrode (SMU2) is measured using the Keithley 4200 semiconductorcharacterization system. The current for both electrodes is held atzero and the resulting potential between the two nodes was measured.(b) A real-time plot of four HCl additions is shown. (c) The surfacepotential from the platinum response is extracted and quantified.The pH response shows high linearity and sensitivity that closelyresembles those stated previously.37
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fig3: Platinum surface potentialresponse. (a) A schematic of the opencircuit potential method is shown. A PDMS well is placed on a platinumsurface and Tris-HCl is added. The on-chip platinum (SMU1) vs thereference electrode (SMU2) is measured using the Keithley 4200 semiconductorcharacterization system. The current for both electrodes is held atzero and the resulting potential between the two nodes was measured.(b) A real-time plot of four HCl additions is shown. (c) The surfacepotential from the platinum response is extracted and quantified.The pH response shows high linearity and sensitivity that closelyresembles those stated previously.37

Mentions: Other earlymethods, such as ion-blocking layers of photoresist or other polymerslike parylene or PVC, have also been demonstrated.32−34 These methodsprimarily rely on a macroscale Ag/AgCl reference electrode for thefluid gate, which is bulky, expensive, and difficult to fabricate.Microfabricated Ag/AgCl electrodes also suffer from potential instabilityand reduced lifetime when submerged in solutions less than 3 M chloride.35 Many examples have utilized a solid-state electrodeas the fluid gate, but each used a differential signal between theISFET and a REFET for pH sensing.15,36 By elucidatingthe platinum pH response in Tris buffer, as seen in Figure 3, this work eliminated the need for a differentialsignal with an ISFET. By blocking the ISFET’s pH response withspotted PVC, the resulting current trace from the ISFET shows thepH response of the platinum. When using the same surface potentialextraction method as the baseline case, the sensor shows the oppositesignal to the addition of NaOH or HCl (Figure 2). This overall pH sensitive system (∼34–36 mV/pH)responds to the change in potential at the platinum fluid electrodeand not the gate dielectric surface potential (see Supporting Information Figure S2). Each case demonstrateda linear relationship between the pH and the REFET response. Futuresteps will focus on metals with demonstrated higher pH sensitivityand more stable response, such as iridium or ruthenium.37 When the QRE platinum electrode is used in conjunctionwith a device without PVC, the surface potential response of the platinumand the gate dielectric mirror each other. This results in a lackof pH sensitivity in the system for this case.


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)

Platinum surface potentialresponse. (a) A schematic of the opencircuit potential method is shown. A PDMS well is placed on a platinumsurface and Tris-HCl is added. The on-chip platinum (SMU1) vs thereference electrode (SMU2) is measured using the Keithley 4200 semiconductorcharacterization system. The current for both electrodes is held atzero and the resulting potential between the two nodes was measured.(b) A real-time plot of four HCl additions is shown. (c) The surfacepotential from the platinum response is extracted and quantified.The pH response shows high linearity and sensitivity that closelyresembles those stated previously.37
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Platinum surface potentialresponse. (a) A schematic of the opencircuit potential method is shown. A PDMS well is placed on a platinumsurface and Tris-HCl is added. The on-chip platinum (SMU1) vs thereference electrode (SMU2) is measured using the Keithley 4200 semiconductorcharacterization system. The current for both electrodes is held atzero and the resulting potential between the two nodes was measured.(b) A real-time plot of four HCl additions is shown. (c) The surfacepotential from the platinum response is extracted and quantified.The pH response shows high linearity and sensitivity that closelyresembles those stated previously.37
Mentions: Other earlymethods, such as ion-blocking layers of photoresist or other polymerslike parylene or PVC, have also been demonstrated.32−34 These methodsprimarily rely on a macroscale Ag/AgCl reference electrode for thefluid gate, which is bulky, expensive, and difficult to fabricate.Microfabricated Ag/AgCl electrodes also suffer from potential instabilityand reduced lifetime when submerged in solutions less than 3 M chloride.35 Many examples have utilized a solid-state electrodeas the fluid gate, but each used a differential signal between theISFET and a REFET for pH sensing.15,36 By elucidatingthe platinum pH response in Tris buffer, as seen in Figure 3, this work eliminated the need for a differentialsignal with an ISFET. By blocking the ISFET’s pH response withspotted PVC, the resulting current trace from the ISFET shows thepH response of the platinum. When using the same surface potentialextraction method as the baseline case, the sensor shows the oppositesignal to the addition of NaOH or HCl (Figure 2). This overall pH sensitive system (∼34–36 mV/pH)responds to the change in potential at the platinum fluid electrodeand not the gate dielectric surface potential (see Supporting Information Figure S2). Each case demonstrateda linear relationship between the pH and the REFET response. Futuresteps will focus on metals with demonstrated higher pH sensitivityand more stable response, such as iridium or ruthenium.37 When the QRE platinum electrode is used in conjunctionwith a device without PVC, the surface potential response of the platinumand the gate dielectric mirror each other. This results in a lackof pH sensitivity in the system for this case.

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