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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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Deviceschematic and characterization (a) A FET/REFET device schematicis shown. Two fluid gate methods are included: A pH-insensitive referenceelectrode and a pH-sensitive on-chip platinum electrode. PVC coversthe left ISFET, rendering it insensitive to pH changes. The rightISFET is left uncovered and is sensitive to pH. The inset plot showstheoretical device response to a hydrogen addition event. The Pt vsFET and RE vs REFET show no overall response to pH changes. Whereasthe Pt vs REFET and RE vs FET show opposite responses. Further detailsare found in Supporting Information FigureS2. (b) An extended gate ISFET is shown. The sensing region consistsof a layer of hafnium oxide on a metal extended gate. (c) One microliterof PVC is spotted on some of the devices to render them pH insensitive,but still functional. (d) A real-time pH response curve of two untreatedISFETs comprising four additions of NaOH followed by four additionsof HCl. The response closely matches the Nernstian response for ISFETgate dielectrics with an average pH response of 54 mV/pH and R2 linearity of 0.995. (e) Quantification ofthe HCl addition steps for multiple devices is shown (n = 16). (f) Real-time response curve for a RE vs FET (black) anda RE vs PVC REFET (red). The PVC-treated device shows minimal pH responseover the duration of the test.
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fig1: Deviceschematic and characterization (a) A FET/REFET device schematicis shown. Two fluid gate methods are included: A pH-insensitive referenceelectrode and a pH-sensitive on-chip platinum electrode. PVC coversthe left ISFET, rendering it insensitive to pH changes. The rightISFET is left uncovered and is sensitive to pH. The inset plot showstheoretical device response to a hydrogen addition event. The Pt vsFET and RE vs REFET show no overall response to pH changes. Whereasthe Pt vs REFET and RE vs FET show opposite responses. Further detailsare found in Supporting Information FigureS2. (b) An extended gate ISFET is shown. The sensing region consistsof a layer of hafnium oxide on a metal extended gate. (c) One microliterof PVC is spotted on some of the devices to render them pH insensitive,but still functional. (d) A real-time pH response curve of two untreatedISFETs comprising four additions of NaOH followed by four additionsof HCl. The response closely matches the Nernstian response for ISFETgate dielectrics with an average pH response of 54 mV/pH and R2 linearity of 0.995. (e) Quantification ofthe HCl addition steps for multiple devices is shown (n = 16). (f) Real-time response curve for a RE vs FET (black) anda RE vs PVC REFET (red). The PVC-treated device shows minimal pH responseover the duration of the test.

Mentions: Electrical detection avoids most ofthese disadvantages by eliminatingthe need for fluorophores and optical detection equipment entirely.To date, systems utilizing ion sensitive field effect transistors(ISFETs),10,11 MOS-capacitors,12 and interdigitatedelectrodes13,14 have demonstrated successfuldetection of nucleic acid amplification. Of these methods, FET arraysoffer benefits of fabrication scalability and row-column addressingfor interrogation of millions of individual devices. The ion-sensitiveFET (ISFET) sensor system replaces the metal gate of a traditionalmetal-on-silicon FET with a fluidic interface and a fluid gate electrode.A potential is applied through the fluid via the electrode to operatethe device and modulate the FET’s source-drain current. Changesin device surface potential through solution pH changes or chargedbiomolecule addition also cause modulations in the ISFET source-draincurrent. In traditional operation, a pH-insensitive reference electrodeholds the fluid gate constant while the source-drain current is monitoredto provide real-time monitoring of solution pH changes. In this paper’sdesign, the changes in surface potential at the ISFET are blockedvia a passivating membrane (polyvinyl chloride). Source-drain currentmodulation occurs via changes in the fluid gate potential by utilizinga pH-sensitive solid-state electrode (see Figure 1a and Supporting Information FigureS-2). Additionally, usage of FETs as biosensors allows scientiststo leverage decades of research and billions of dollars in investmentin computer chip processing/fabrication to expedite the developmentprocess. Sophisticated semiconductor fabrication foundries offeredby companies such as Taiwan Semiconductor Manufacturing Company (TSMC)can manufacture devices with high yield, near-ideal and highly repeatabledevice characteristics with huge amenability for scale-up. For anexcellent overview of ISFET operation, sensing modes, and importantexperimental design factors, the reader is referred to a review byP. Bergveld.15


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)

Deviceschematic and characterization (a) A FET/REFET device schematicis shown. Two fluid gate methods are included: A pH-insensitive referenceelectrode and a pH-sensitive on-chip platinum electrode. PVC coversthe left ISFET, rendering it insensitive to pH changes. The rightISFET is left uncovered and is sensitive to pH. The inset plot showstheoretical device response to a hydrogen addition event. The Pt vsFET and RE vs REFET show no overall response to pH changes. Whereasthe Pt vs REFET and RE vs FET show opposite responses. Further detailsare found in Supporting Information FigureS2. (b) An extended gate ISFET is shown. The sensing region consistsof a layer of hafnium oxide on a metal extended gate. (c) One microliterof PVC is spotted on some of the devices to render them pH insensitive,but still functional. (d) A real-time pH response curve of two untreatedISFETs comprising four additions of NaOH followed by four additionsof HCl. The response closely matches the Nernstian response for ISFETgate dielectrics with an average pH response of 54 mV/pH and R2 linearity of 0.995. (e) Quantification ofthe HCl addition steps for multiple devices is shown (n = 16). (f) Real-time response curve for a RE vs FET (black) anda RE vs PVC REFET (red). The PVC-treated device shows minimal pH responseover the duration of the test.
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fig1: Deviceschematic and characterization (a) A FET/REFET device schematicis shown. Two fluid gate methods are included: A pH-insensitive referenceelectrode and a pH-sensitive on-chip platinum electrode. PVC coversthe left ISFET, rendering it insensitive to pH changes. The rightISFET is left uncovered and is sensitive to pH. The inset plot showstheoretical device response to a hydrogen addition event. The Pt vsFET and RE vs REFET show no overall response to pH changes. Whereasthe Pt vs REFET and RE vs FET show opposite responses. Further detailsare found in Supporting Information FigureS2. (b) An extended gate ISFET is shown. The sensing region consistsof a layer of hafnium oxide on a metal extended gate. (c) One microliterof PVC is spotted on some of the devices to render them pH insensitive,but still functional. (d) A real-time pH response curve of two untreatedISFETs comprising four additions of NaOH followed by four additionsof HCl. The response closely matches the Nernstian response for ISFETgate dielectrics with an average pH response of 54 mV/pH and R2 linearity of 0.995. (e) Quantification ofthe HCl addition steps for multiple devices is shown (n = 16). (f) Real-time response curve for a RE vs FET (black) anda RE vs PVC REFET (red). The PVC-treated device shows minimal pH responseover the duration of the test.
Mentions: Electrical detection avoids most ofthese disadvantages by eliminatingthe need for fluorophores and optical detection equipment entirely.To date, systems utilizing ion sensitive field effect transistors(ISFETs),10,11 MOS-capacitors,12 and interdigitatedelectrodes13,14 have demonstrated successfuldetection of nucleic acid amplification. Of these methods, FET arraysoffer benefits of fabrication scalability and row-column addressingfor interrogation of millions of individual devices. The ion-sensitiveFET (ISFET) sensor system replaces the metal gate of a traditionalmetal-on-silicon FET with a fluidic interface and a fluid gate electrode.A potential is applied through the fluid via the electrode to operatethe device and modulate the FET’s source-drain current. Changesin device surface potential through solution pH changes or chargedbiomolecule addition also cause modulations in the ISFET source-draincurrent. In traditional operation, a pH-insensitive reference electrodeholds the fluid gate constant while the source-drain current is monitoredto provide real-time monitoring of solution pH changes. In this paper’sdesign, the changes in surface potential at the ISFET are blockedvia a passivating membrane (polyvinyl chloride). Source-drain currentmodulation occurs via changes in the fluid gate potential by utilizinga pH-sensitive solid-state electrode (see Figure 1a and Supporting Information FigureS-2). Additionally, usage of FETs as biosensors allows scientiststo leverage decades of research and billions of dollars in investmentin computer chip processing/fabrication to expedite the developmentprocess. Sophisticated semiconductor fabrication foundries offeredby companies such as Taiwan Semiconductor Manufacturing Company (TSMC)can manufacture devices with high yield, near-ideal and highly repeatabledevice characteristics with huge amenability for scale-up. For anexcellent overview of ISFET operation, sensing modes, and importantexperimental design factors, the reader is referred to a review byP. Bergveld.15

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