A new approach to immunoFET operation

TLDR: A new detection method is presented for the detection of an immunological reaction taking place in a membrane, which covers the gate area of an ISFET, which gives direct information about specific charge properties of the macromolecules to be studied.

About: This article is published in Biosensors and Bioelectronics.The article was published on 1990-01-01 and is currently open access. It has received 149 citations till now. The article focuses on the topics: Isoelectric point & Membrane.

Frequently asked questions

Q1. What are the contributions in "A new approach to immunofet operation" ?

A new method is presented for the detection of an immunological reaction taking place in a membrane, which covers the gate area of an ISFET. Up to now, the authors have studied the membrane adsorption of lysozyme, human serum albumin ( HSA ) and the immune reaction of HSA with the antibody anti-human serum albumin ( cxHSA ). The simple procedure of one incubation stage followed by one detection stage, without separate washing and labelling techniques, gives direct information about specific charge properties of the macromolecules to be studied. By stepwise changing the electrolyte concentration of the sample solution, a transient diffusion of ions through the membrane-protein layer occurs, resulting in a transient membrane potential, which is measured by the ISFET. No membrane potential is induced at zero fixed charge density as occurs at a protein characteristic pH. Isoelectric points of embedded proteins can be determined by detecting the zero potential response. 

Q2. What is the aHSA concentration in the membrane?

The membrane becomes less porous during the adsorption of the large aHSA molecules: the membrane clogs, so a concentration gradient in the membrane will occur. 

Q3. What is the way to measure the charge density in the membrane?

In order to measure the charge density in the membrane, the authors must select an ion combination which is appropriate for carrying out the experiments. 

Q4. What is the amplitude of the transient membrane potential?

- Apparently, the amplitude of the transient membrane potential is dependent on the sum of any fixed charges present in the bare membrane including those of adsorbed proteins. 

Q5. What is the importance of a membrane covered ISFET?

It is very important that no permselectivity exists, implying that atequilibrium only true pH values are measured with the membrane covered ISFET. 

Q6. How can lysozyme be incorporated into the membrane?

Lysozyme can be incorporated into the membrane by means of two preparation methods:1. Lysozyme can be mixed with polystyrene/agarose prior to ISFET coating. 

Q7. What was the amplitude of the transient after the electrolyte step decrease?

2. Since the ISFET with the membrane was exposed to the unbuffered 40 mM KC1 solution only for a short time without reaching equilibrium, the amplitude of the transient after the electrolyte step decrease was not important. 

Q8. Why is the detection limit lower than that found for ISFETs prepared following preparation method 1?

The lower detection limit, as compared to that found for ISFETs prepared following preparation method 1, is due to the fact that protein accumulation in the membrane can occur. 

Q9. What is the aHSA concentration of the polyclonal serum?

The aHSA concentration of the polyclonal serum was defined as the titre at which full precipitate formation took place with an equimolar concentration of HSA. 

Q10. What was the thickness of the ISFETs used in the experiments?

The ISFETs used in the experiments were n-channel ISFETs with a 70 nm thermally grown SiOZ and 120 nm Ta205 gate, which were connected to a source and drain follower (Bergveld, 1981). 

Q11. Why is the hinge point of a protein different from the one of lysozy?

Due to less charge contribution per HSA molecule, the shift of the inversion point with the HSA concentration is relatively small, compared to that for lysozyme. 

Q12. How did the lysozyme concentrations in the membrane after the preparation be determined?

The concentrations of lysozyme in the membrane after the preparation were 0,2 f 1,6 f 1,lO + 2,20 + 2,40 A 4,60 + 4,85 f 5 PM, respectively. 

Q13. What is the main quantity determining the detection limit of the present method?

The experiments corroborate the assumption that the protein charge density near pIO is the main quantity determining the detection limit of the present method. 

Q14. How is the detection of HSA inversion points with different devices?

The reproducibility of the detection of the HSA inversion points with different devices measured for a concentration of HSA, is within + 0.05 pH.5.5