In this paper, we describe a novel biosensor for the detection of alanine aminotransferase ALT, EC 2.
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ALT is found mainly in the liver, but is also found in red blood cells, heart cells, muscle tissue and other organs, such as the pancreas and kidneys  , . Serum ALT levels are an indicator for liver damage and its detection is considered the gold standard biomarker of hepatotoxicity  ,  , . Most ALT biosensors are based on the detection of the enzymatic activity of the ALT enzyme as opposed to the detection of the protein itself  ,  ,  , .
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In this article we present the selection of ALT binding peptides from an M13 phage displayed peptide library and the characterization of the binding affinities of the peptides. Once the most promising peptide was selected, a cysteine-modified free peptide was synthesized and transferred to the biosensor platform. The general approach Figure 1 can be extended to develop biosensors for a wide variety of target analytes.
The methodology can be easily applied in a relatively short period of time at low cost. All reagents used were analytical grade or higher unless otherwise stated.
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Louis, Missouri. Louis, MO. All solutions were prepared using Milli-Q water. ALT 2. The well surfaces were then blocked with blocking buffer 0. The Ph. Wells were washed 10 times with TBST to remove unbound phage. The eluted phage were amplified in E. This biopanning process was repeated for five rounds. The concentration of Tween 20 in the washing step was increased from 0. After the fifth round of selection, twelve blue monocolonies were randomly picked and amplified for DNA sequencing. Microplates were coated with ALT 2. Varying amounts of amplified phage clones were added to each well and incubated for 1 hr at room temperature.
In order to estimate the apparent dissociation constant for the ALT clone, further ELISA experiments were performed with extended phage concentrations. ALT enzyme kinetics were measured to determine the impact of the ALT peptide on the activity of the enzyme. The kinetic rates were fit to the bi-bi ping-pong mechanism Eqn.
Free ALT peptide inhibitor I was added to the reactions at concentrations from 0—0. The rates plotted on a Lineweaver-Burk plot to understand the mode of inhibition and the rates were also fit to the bi-bi ping-pong equation with a competitive inhibitor Eqn. The operation of the setup was described previously .
After a stable baseline was obtained in buffer, a peptide solution in the buffer was flowed to the cell. Usually an abrupt change in the signal occurs with the valve switching. In about 5 min, the new solution arrived at the cell and caused a frequency change due to peptide binding to the crystal surface. After the binding reached a steady state, buffer rinse was then applied and no further change in frequency was observed.
Similar operations were performed for the addition of the cysteine and ALT solutions, as well as solutions of other protein competitors such as BSA and SA. All experiments were repeated three times and similar results were obtained. A gold wire dia. CV and EIS were performed as previously described . Briefly, the gold electrodes were prepared in 0. Impedance spectra were recorded over a frequency range of 0. A single sinusoidal AC voltage of 10 mV was superimposed on the open-circuit potential typically 0.
The impedance was recorded and a Nyquist plot was obtained.
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A Randles circuit was used to fit the semi circular curves in order to calculate the charge transfer resistance R CT. A large commercially available phage display library was biopanned over the immobilized ALT target protein. The yield of phage binding to the plate increased each round after the second Table S1 , even as the stringency of the selections was increased by the addition of Tween All measurements were performed in triplicate and error bars represent standard deviations.
Since the phage particles have approximately 5 copies of the peptide per particle, it is possible that the true affinity of the free peptide for the ALT protein will be different from the apparent value due to the loss of the avidity effect. The ALT target protein has a large surface area for peptide binding and the binding of the peptide in or near the active site of the enzyme could possibly affect the catalytic properties of the enzyme.
Therefore, the impact of the peptide on the kinetics of the ALT enzyme was investigated. In the absence of the peptide, the kinetic data fit well to the bi-bi ping-pong equation Eqn. Lineweaver-Burk plots of pyruvate production at varying alanine concentrations are shown in Figure 4. The data suggests that the ALT binding peptide is a competitive inhibitor for the active site, with respect to alanine.
The data were fit to the bi-bi ping-pong mechanism with competitive inhibition Eqn. All data were collected in triplicate and R 2 values are shown for the best fit line for each inhibitor concentration. In order to use the phage selected ALT peptide in the development of an ALT biosensor, the peptide was modified with a C-terminal cysteine for immobilization on gold. The preparation of the sensor consisted of two steps: the immobilization of the peptide and blocking of empty sites with free cysteine.
The abrupt changes in the QCM noted by arrows Figure 5A are due to pressure changes caused by valve switching. This phenomenon was observed in all the experiments. All the experiments were repeated three times with similar results each time. All three techniques show the successful immobilization of the peptides on the gold surface. Backfilling of the surface cysteine did not cause any mass change in QCM since both the amount and molecular weight of the blocking cysteine is small. However, the backfilling did result in a current increase and a prominent decrease in the impedance signal smaller semi-circles.
A similar effect was observed in our previous studies  and in studies of other modified surfaces . The impedance decrease upon backfilling can be explained by the easier charge transfer through tunneling across the shorter cysteine. The surface density of a similar sized troponin-binding peptide immobilized on a gold surface has previously been reported to be 7. Inamori et al. A the change in frequency during the immobilization of peptide Pep, 0. The same techniques were used to demonstrate the affinity of the immobilized peptide for ALT, as shown in Figure 6.
No change was observed for the buffer control in all three cases. All three techniques indicate binding of ALT to the immobilized peptide. To begin to study the selectivity of the peptide, similar experiments were performed with streptavidin SA and bovine serum albumin BSA. The small frequency change seen with the control proteins might be due to nonspecific binding of SA and BSA with unoccupied sites on the surface or non-specific interactions with the peptide. Although all three techniques demonstrate a signal change upon the binding of ALT, the CV technique is a poor candidate for quantitative measurement since the detection range is limited by the fact that the current decreases upon target binding.
The sensitivity of the QCM system was 8. Each run was performed using a single electrode with successive tests in ALT solutions from low to high concentration. Error bars represent standard deviations obtained from triplicate measurements. Based on the QCM response curve, the kinetic binding constants can be deduced. Our overall goal is to develop a streamlined platform for the rapid development of new biosensors that can be used to detect virtually any desired protein target.
Phage display is a commonly used method for identifying new binding motifs, and commercially available kits make this process broadly accessible  , . Biopanning procedures are well documented, and new binding peptides can usually be identified in a short period of time 1—2 weeks. There are several detection methods available that can be used to create a biosensor from a recognition peptide.
We have chosen electrochemical techniques since they are generally simple, inexpensive, and can be readily incorporated into microfluidic devices. In this manuscript, we demonstrate this process from start to finish to create a new biosensor for the detection of ALT. ALT is an enzyme, and one approach for its detection is to monitor its enzymatic activity  ,  , .
This approach can be very sensitive, but it is not a general platform for biosensor development, and this approach needs to be redeveloped for each new enzyme to be detected. We have chosen instead to directly detect the presence of the protein using an evolved binding peptide, and in this way the detection method is generic and does not depend on the enzymatic activity of the target protein.
The phage display library converged to a new binding sequence after 5 rounds of biopanning ALT and this peptide exhibited nanomolar affinity for ALT when displayed on phage particles. We further explored whether the peptide was binding near the active site of the enzyme through enzyme inhibition studies. The peptide was found to be a competitive inhibitor for ALT with a nanomolar inhibition constant.
Therefore, it is possible that the enzyme is still active in the bound state, and future work will be necessary to determine if a biosensor can be created based on detecting the bound ALT activity instead of monitoring the mass change that occurs upon binding of the enzyme. Another concern with this approach is that peptides identified using phage display may lose affinity for their target when they are displayed on a different platform.
There are, on average, 5 copies of the ALT peptide on the surface of the M13 phage, and this avidity effect gives rise to an apparent nanomolar dissociation constant. This proved not be a significant problem as, when peptides were immobilized on a solid surface, a true dissociation constant for the ALT binding peptide remained in the nanomolar scale range. As we previously found with Troponin I, EIS appears to be the optimal method for the electrochemical detection of the bound target . The procedure is straightforward, and high sensitivities and low LOD values are obtained.
The only downside to the EIS technique is the need to measure the impedance signal in the presence of a redox couple after the sample has been exposed to the peptide sensing layer. But, the EIS technique produces a larger linear sensing range than can be obtained with QCM and it is more robust than QCM as it is less sensitive to environmental perturbations.
Although we have demonstrated that the ALT peptide is selective for ALT as compared to BSA and SA, further work will be required to explore the performance of the biosensor in real samples such as tissue culture media or plasma, and in the presence of other potential interferents. Although this approach will require further development before it can be used to create a biosensor for clinical use, the use of unstructured peptides obtained via phage display coupled with electrochemical detection methods has several advantages compared to other biosensor platforms described in the literature.
Alternative recognition elements such as antibodies are time consuming and expensive to produce, and aptamer-based sensors can require complex sample labeling and modification techniques . The unstructured peptides used in this approach can be easily selected against virtually any target and are inexpensive to synthesize, but they may suffer from poor selectivity in complex solutions.
In summary, we have developed a general method for the creation of new biosensors using short synthetic peptides obtained via biopanning of a phage displayed library. The whole process includes: target selection and immobilization, phage display to select binding peptides, peptide synthesis with a terminal thiol, QCM in-situ monitoring of peptide immobilization, and sensor detection using electrochemical techniques Figure 1.
We have demonstrated this approach by creating a new biosensor for a well-known biomarker for hepatocellular toxicity, ALT. The new biosensor had a LOD value just below the physiological concentration of the target protein in human blood. Since both phage display and EIS are widely used techniques, this general approach to biosensor development is straightforward and can be readily applied for the development of biosensors to detect almost any desired protein target.
Uninhibited ALT enzyme kinetic data. All data were collected in triplicate and error bars represent standard errors. Enrichments obtained by selecting the phage displayed peptide library over immobilized ALT. The authors would like to thank Ms. Victoria Sun for assistance with the collection of the enzyme kinetic data. We also thank Mr. Oren Shur, Dr.
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Elliot Campbell, and Ms. Asli Sahin for thoughtful reviews of the manuscript. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract There is a consistent demand for new biosensors for the detection of protein targets, and a systematic method for the rapid development of new sensors is needed.
Willson, University of Houston, United States of America Received: March 14, ; Accepted: August 24, ; Published: October 7, This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
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