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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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pH-LAMP optimization. (a) The detection limit of a commerciallyavailable E. coli O157:H7 kit is shown. LAMP allowsfor detection of 100–1000 CFU/reaction in less than 30 min.The inset graph provides the normalized real-time amplification data.(b) The pH-LAMP detection limit is shown. Regardless of starting E. coli concentration, the resulting pH change is consistentat around −0.2 pH units. (c) Reducing the Tris-HCl buffer concentrationin the reaction mix increases the pH change associated with amplification.The maximum pH change observed in these tests was −1.2 pH units.(d) Decreasing the isothermal amplification buffer concentration alsoreduced the ionic strength of the solution. This requires the reactiontemperature to be reduced to achieve consistent threshold times. Thresholdtimes were consistent down to 8–12 mM Tris-HCl before significantincreases in threshold time are observed.
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fig4: pH-LAMP optimization. (a) The detection limit of a commerciallyavailable E. coli O157:H7 kit is shown. LAMP allowsfor detection of 100–1000 CFU/reaction in less than 30 min.The inset graph provides the normalized real-time amplification data.(b) The pH-LAMP detection limit is shown. Regardless of starting E. coli concentration, the resulting pH change is consistentat around −0.2 pH units. (c) Reducing the Tris-HCl buffer concentrationin the reaction mix increases the pH change associated with amplification.The maximum pH change observed in these tests was −1.2 pH units.(d) Decreasing the isothermal amplification buffer concentration alsoreduced the ionic strength of the solution. This requires the reactiontemperature to be reduced to achieve consistent threshold times. Thresholdtimes were consistent down to 8–12 mM Tris-HCl before significantincreases in threshold time are observed.

Mentions: Loop-mediated isothermalamplification(LAMP) was developed in the early 2000s as an isothermal alternativeto PCR.40,41 LAMP utilizes four distinct primers recognizingsix regions of a targeted gene. This method offers high sensitivity(shown in Figure 4a) and superior specificityto PCR, which enables an inherently nonspecific method of amplificationdetection, such as an intercalating dye or, in this case, pH detection.Additionally, LAMP provides a yield of >500 μg/mL of DNA.PCR,on the other hand, only offers a maximum yield of around 40 μg/mL.41 The level of DNA generation in LAMP resultsin a higher potential pH change for a given buffer concentration.Theoretically, in standard buffered amplification solution of 20 mMTris-HCl, LAMP will yield a pH change of ∼−0.136 units,whereas PCR will only produce a pH change of ∼-0.01 units.(See Supporting Information for a descriptionof the pH change calculations.) As shown in Figure 4b, using the Eiken kit for E. coli O157:H7,the LAMP reaction generates a pH change ranging from −0.15to −0.20 pH units. This change is consistent across the entirerange of starting template concentration since each reaction was allowedto run to completion.


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)

pH-LAMP optimization. (a) The detection limit of a commerciallyavailable E. coli O157:H7 kit is shown. LAMP allowsfor detection of 100–1000 CFU/reaction in less than 30 min.The inset graph provides the normalized real-time amplification data.(b) The pH-LAMP detection limit is shown. Regardless of starting E. coli concentration, the resulting pH change is consistentat around −0.2 pH units. (c) Reducing the Tris-HCl buffer concentrationin the reaction mix increases the pH change associated with amplification.The maximum pH change observed in these tests was −1.2 pH units.(d) Decreasing the isothermal amplification buffer concentration alsoreduced the ionic strength of the solution. This requires the reactiontemperature to be reduced to achieve consistent threshold times. Thresholdtimes were consistent down to 8–12 mM Tris-HCl before significantincreases in threshold time are observed.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: pH-LAMP optimization. (a) The detection limit of a commerciallyavailable E. coli O157:H7 kit is shown. LAMP allowsfor detection of 100–1000 CFU/reaction in less than 30 min.The inset graph provides the normalized real-time amplification data.(b) The pH-LAMP detection limit is shown. Regardless of starting E. coli concentration, the resulting pH change is consistentat around −0.2 pH units. (c) Reducing the Tris-HCl buffer concentrationin the reaction mix increases the pH change associated with amplification.The maximum pH change observed in these tests was −1.2 pH units.(d) Decreasing the isothermal amplification buffer concentration alsoreduced the ionic strength of the solution. This requires the reactiontemperature to be reduced to achieve consistent threshold times. Thresholdtimes were consistent down to 8–12 mM Tris-HCl before significantincreases in threshold time are observed.
Mentions: Loop-mediated isothermalamplification(LAMP) was developed in the early 2000s as an isothermal alternativeto PCR.40,41 LAMP utilizes four distinct primers recognizingsix regions of a targeted gene. This method offers high sensitivity(shown in Figure 4a) and superior specificityto PCR, which enables an inherently nonspecific method of amplificationdetection, such as an intercalating dye or, in this case, pH detection.Additionally, LAMP provides a yield of >500 μg/mL of DNA.PCR,on the other hand, only offers a maximum yield of around 40 μg/mL.41 The level of DNA generation in LAMP resultsin a higher potential pH change for a given buffer concentration.Theoretically, in standard buffered amplification solution of 20 mMTris-HCl, LAMP will yield a pH change of ∼−0.136 units,whereas PCR will only produce a pH change of ∼-0.01 units.(See Supporting Information for a descriptionof the pH change calculations.) As shown in Figure 4b, using the Eiken kit for E. coli O157:H7,the LAMP reaction generates a pH change ranging from −0.15to −0.20 pH units. This change is consistent across the entirerange of starting template concentration since each reaction was allowedto run to completion.

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