Biosensing with Virus Electrode Hybrids

Virus electrodes address two major challenges associated with biosensing. First, the surface of the viruses can be readily tailored for specific, high affinity binding to targeted biomarkers. Second, the viruses are entrapped in a conducting polymer for electrical resistance‐based, quantitative measurement of biomarker concentration. To further enhance device sensitivity, two different ligands can be attached to the virus surface, and increase the apparent affinity for the biomarker. In the example presented here, the two ligands bind to the analyte in a bidentate binding mode with a chelate‐based avidity effect, and result in a 100 pM experimentally observed limit of detection for the cancer biomarker prostate‐specific membrane antigen. The approach does not require enzymatic amplification, and allows reagent‐free, real‐time measurements. This article presents general protocols for the development of such biosensors with modified viruses for the enhanced detection of arbitrary target proteins. © 2015 by John Wiley & Sons, Inc.


INTRODUCTION
This article describes a general protocol for the development of a biosensor aimed at efficiently detecting cancer biomarkers in a patient's biological fluids, including blood and urine. The virus electrode biosensors provide highly sensitive, reagent-free, real-time detection of the targeted protein (Mohan et al., 2013). M13 filamentous bacteriophage, the biological recognition element, display peptide ligands to a specific biomarker. These viruses and their phage-displayed ligands are incorporated into the biosensor during electrochemical polymerization with a conducting organic polymer, poly-3,4ethylenedioxythiophene (PEDOT) onto gold electrodes (Yang et al., 2006(Yang et al., , 2008bWeiss and Penner, 2008;Diaz et al., 2009;Arter et al., 2010). Upon binding, a perturbation in the electrode's electrical impedance results in a quantitative measurement of the target biomarker. In this article, the target analyte is prostate-specific membrane antigen (PSMA), a prostate cancer biomarker (Murphy et al., 1998;Schülke et al., 2003;Chuang et al., 2007). wrapping around the phage surface. The dual display of ligands results in a bidentate binder, and enhanced PSMA detection from both a dense concentration of ligands and a chelate-based, avidity effect (Murase et al., 2003). Biosensing with virus-PEDOT films provided a 100 pM limit of detection (LOD) for PSMA in synthetic urine without requiring enzymatic or other amplification (Mohan et al., 2013). The reported approach leverages two ligands with varying target affinities to achieve high sensitivity for the targeted biomarker.
Phage-based biosensors offer a number of key advantages. First, phage and the displayed peptide ligands are extraordinarily stable (Kay et al., 1996). For example, similar virushybrid surfaces are stable for over 14 hr in rapidly flowing, high ionic strength buffer (Yang et al., 2008b), and the phage retain their binding abilities after 6 weeks at 65°C (Brigati and Petrenko, 2005). Second, phage form liquid crystals at high concentration, which can maximize the density of packing in the biosensors. We have observed this dense packing, which causes the phage to line-up like match sticks, in atomic force microscopy of our covalent virus surface (Yang et al., 2008b). Third, the "kelp forest architecture" of the covalent virus surface, which we observed by quartz crystal microbalance (QCM) allows rapid kinetics in binding to biomarkers (Yang et al., 2008a). Such multi-point, cooperative binding has been observed by surface plasmon resonance (SPR) for a phagebased surface (Nanduri et al., 2007).
Most importantly, phage binding can be readily adapted for the recognition of arbitrary molecular targets. Phage display has been successfully applied to a wide range of targets, including proteins (as described here), DNA, and small molecules. The architecture described here, for example, can be applied to the detection of different biomarkers for a broad range of diseases simply by modifying the viral DNA to display ligands targeting biomarkers associated with each disease. et al., 2000). Thus, it serves as a suitable target for non-invasive testing in urine from patients. Phage-displayed peptide libraries were used to select for ligands that selectively bind to PSMA (Kehoe and Kay, 2005;Levin and Weiss, 2006;Arter et al., 2012). Theoretically, the ligands displayed on phage could either be peptides or proteins, but for the secondary recognition ligands, peptides are preferred for dense ligand display. In addition, the high copy number of peptide ligands on the phage surfaces increases their effective concentration in the bioaffinity layer.
A wide range of bioorthogonal chemistries could allow attachment of the two halves of the secondary recognition ligand. A modular approach is preferred rather than the synthesis of a long peptide to obtain higher yields of the synthetic peptide. When using this approach, the oligolysine half of the secondary recognition ligand can remain constant for many different peptide ligands wrapped onto the phage. Thus, it is advantageous to synthesize the two halves separately to maximize the flexibility of the experimental design and efficiency. The copper-catalyzed azide-alkyne cycloaddition, a 'click' reaction, can link the two halves of the wrapper, as the reaction offers a convergent synthesis that proceeds at room temperature in aqueous solution (Rostovtsev et al., 2002).
The bioaffinity matrix can be affixed to the working electrode of the electrochemical biosensor by adsorption, entrapment, a specific interaction, and covalent attachment. Biomolecular entrapment of the viruses within a conducting polymer matrix provides robust attachment and allows the ligands to remain functional for the recognition of the target molecules (Cosnier, 1999). Additionally, the method is expeditious, and avoids potential phage-degrading steps.
The protocols in this article present a general method for the detection of an arbitrary disease biomarker, termed a target molecule, using electrochemical biosensors consisting of phage-display ligands wrapped with additional secondary recognition ligands to increase the biosensor's sensitivity for the target molecule (see Fig. 2). Three basic procedures are described. First, the secondary recognition ligands are synthesized from individual peptides. Second, phage-displayed ligands and control phage are propagated and isolated. Third, the virus-PEDOT films are formed before generation of a calibration curve for target protein detection. As a specific example to illustrate these steps, the incorporation of peptide ligands into the bioaffinity layer for the detection of PSMA is described here. Figure 3 PSMA ligands, sequence and nomenclature. The "X" in the structure describes the entries in the first column, and is the PSMA binding ligand; K 14 is defined as a peptide composed of 14 lysines.

Peptide synthesis of ligands
Conventional solid phase peptide synthesis was used for the generation of alkyne-and azide-functionalized peptides (Fig. 3). Next, the peptides were purified by reverse-phase HPLC, and the fractions characterized by matrix-assisted laser desorption/ionization (MALDI). The relevant fractions were then combined, and their purity analyzed by analytical reverse-phase HPLC.

CLICK CHEMISTRY REACTION: PREPARATION OF K CS -1 AND K CS -2
After the azide-and alkyne-functionalized peptides have been synthesized and purified, the next step is to link them by the click reaction shown in Figure 4. The resultant ligands are listed as K CS -1 and K CS -2 in Figure 3. The reaction protocol for the click reaction is modified from the Lumiprobe protocol for oligonucleotides (see Internet Resources section). While working with Cu(I), the disproportionation of Cu(I) to Cu(II) or Cu(0) leads to the generation of the non-catalytic Cu states. Ligands such as tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) are commonly used to stabilize the Cu(I) state. Alternatively, if the reaction proceeds at a rapid rate, the addition of ligands can be avoided. Triethylammonium acetate acts as a buffer.

Biosensing with Virus Electrode Hybrids
Setting up the cycloaddition reaction 1. Prepare a 5 mM ascorbic acid solution in HPLC-grade water.
The solution is unstable and should be discarded after each use. HPLC-grade water is recommended, but not required; inconsistent results were observed from reactions performed in Milli-Q water.
The concentration of both azide-and alkyne-functionalized peptides in the final reaction mixture will be 40 μM. Higher concentrations resulted in lower yields. Parallel 40 μM, 1-ml scale reactions should be used to obtain higher yields and more product. The reaction mixtures from the parallel reactions can then be combined before characterization of the reaction product.
3. Add 50 μl triethylammonium acetate buffer solution to a final concentration of 50 mM.
4. Add required amount of HPLC-grade water such that the final reaction volume after the addition of solutions below will be an estimated 1 ml. Vortex solution.
If necessary, the azide-functionalized peptide may be used in slight excess as suggested in the Lumiprobe protocol. A 1:1 ratio provided the expected results with the peptides used here.
6. Sparge reaction mixture by bubbling an inert gas (e.g., nitrogen) through the mixture for 30 sec.
The reaction vessel (conical tube) should be sealed immediately to avoid absorption of oxygen by the solution.
A precipitate observed after addition of the reagents likely results from a high concentration of azide-functionalized peptide ligand. In addition to decreased concentrations of the azide-functionalized peptide ligand, the solution can be heated to 80°C for a few minutes to attempt resolubilization.
Characterize product formation 11. Combine reactions into one conical tube.
12. Run matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry on a sample of the reaction product to confirm product formation.
α-Cyano-4-hydroxycinnamic acid was used as the matrix for sample preparation.
Purify the cycloaddition reaction product 13. Concentrate reaction mixture using 2 kD molecular weight cut-off micro concentrator.
In general, ten reactions were run and concentrated to 500 to 1000 μl for further purification.
The product was purified using multiple runs of analytical scale HPLC. Fractions were analyzed by MALDI-TOF, and the appropriate fractions from each run were combined.
15. Place fractions under speed vacuum to remove solvent.
HPLC-grade water was used for the final resuspension. The percentage of acetonitrile should be used as needed for solubility.

PHAGE PROPAGATION, ISOLATION, PURIFICATION, AND QUANTIFICATION
M13 bacteriophages infect Gram-negative bacteria (e.g., Escherichia coli), and can be readily isolated from bacterial culture. Within the encapsulated phagemids (plasmid-like vectors), open reading frames (ORFs) encode peptide ligands as fusions to a periplasmic localization signal sequence (stII leader sequence: MKKNIAFLLASMFVFSIATNAYA) and the N-terminus of P8, the major coat protein for fusion on the phage surface. The signal peptide directs the resultant protein into the periplasm. Furthermore, the stII leader sequence also incorporates a signal peptidase site, which is cleaved off leading to the displayed peptide fused to the N-terminus of the P8 coat protein through the Gly-Ser linker (GGGSGSSSGGGSGGG). The order of the resultant fusion is stII leader sequence-(N terminus-peptide ligand)-(Gly-Ser linker)-P8-COOH. DNA encoding displayed peptides were introduced by mutagenesis (Smith, 1985). The phagemid applied below also includes an antibiotic-resistance marker to allow selections for its propagation in the presence of carbenicillin. Other proteins required for phage propagation and assembly are separately provided by co-infecting the cultures with helper phage (M13 KO7). The genome of KO7 has a mutated packaging signal, which decreases its efficiency and ensures preferential packaging of the phagemid DNA. The encapsulated viruses are then secreted from the bacteria, and can be precipitated from the culture using polyethylene glycol (PEG)-sodium chloride (NaCl) precipitation. This protocol details all steps necessary to obtain purified phage.

Starter cultures for phage propagation
3. Aliquot 2 ml of 2YT medium into each of two 15-ml Falcon tubes, and add 2 μl carbenicillin and 1 μl tetracycline from the antibiotic stocks to each tube.
XL1 Blue E. coli carry a plasmid with genes encoding tetracycline resistance and F pili, which are required for phage infection.
4. Pick small quantities from the center of two individual colonies from step 2 to add to the two tubes prepared in step 3 (one colony transfer per tube).
Shaking the cultures with the tubes in a slanted position improves growth as it provides increased aeration. Growing the cultures to log phase is crucial to obtain high infection efficiency (Lowman and Clackson, 2004 12. Transfer overnight culture to another 250-ml centrifuge tube. Centrifuge at 15,344 × g for 10 min at 4°C. 13. Transfer supernatant from step 12 to the centrifuge tube containing PEG-NaCl (step 11). Mix by inverting the tube ten times.
Care should be taken during transfer to avoid disturbing the cell pellet.
14. Incubate solution on ice for 1 hr. 17. Remove excess supernatant by blotting the centrifuge tubes on paper towels for 1 to 3 min.
This step pelletizes the insoluble debris from the cell culture.
Perform second precipitation 20. Transfer supernatant to a fresh centrifuge tube. Add a one-fifth volume of PEG-NaCl, and incubate on ice for 1 hr. 23. Resuspend phage pellet in 12 mM LiClO 4 and re-centrifuge at 13,776 × g for 10 min at 4°C.

BIOSENSING: FORMATION OF THE BIOAFFINITY MATRIX AND ELECTROCHEMICAL DETECTION
After the completion of Basic Protocols 1 and 2, the bioaffinity matrix of the biosensor is prepared for electrochemical detection of PSMA. Incubation and binding of the target analyte, PSMA, to the biological recognition element produces a change in its electrical properties. Specifically, an increase in biosensor resistance is observed upon PSMA binding. As described above, PEDOT is formed by the electrochemical polymerization of EDOT in the presence of LiClO 4 and virus particles. The negative charge on the virus surface drives phage incorporation into the positively charged PEDOT polymer, forming through electrodeposition onto the gold electrode. The concentration of viruses used in the EDOT-LiClO 4 solution is an important parameter. Increased virus concentration leads to higher incorporation of viruses into the virus-PEDOT film . A 3 nM virus solution provided an optimal working concentration. The sensitivity for PSMA detection was further improved by wrapping the incorporated viruses with additional secondary recognition ligands.
The calibration curve provides the relative changes in resistance obtained over a wide range of PSMA concentrations. Data were collected over a wide range of frequencies and analyte concentrations to determine the optimal frequency to use. For the calibration curve reported previously, the calculations performed and parameters were extracted from the 1000-Hz data (Donavan et al., 2011). As a starting point for measuring a calibration curve, the target concentration should be well above the limit of detection (LOD) for the device. The negative controls must examine non-specific binding between the target and the components forming the bioaffinity matrix; measurements with target binding to PEDOT films (no virus) provide an important control. Such controls also must test whether wrapping the phage with additional ligands increases resistance and non-specific binding. The data obtained can be analyzed to compute parameters such as the apparent K d for the interaction between the target and the virus-displayed ligands, the LOD, and potential cooperativity. Physically clean the electrode 5. Polish a single gold 3-mm diameter electrode (reusable). Cut a sheet of micropolishing cloth into smaller pieces ß5.1 cm × 5.1 cm (2 inches × 2 inches). Attach a piece to a work bench, using the adhesive on the reverse surface. Place a small quantity of 1-μm diamond polishing paste on one corner.

Materials
To avoid additional variables, the same electrode was used for the reported measurements.
6. With some paste on the electrode surface, move the electrode in circles on the polishing cloth approximately ten times, then apply more paste and slightly rotate

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Volume 7 Current Protocols in Chemical Biology the electrode before repeating the polishing step at a fresh spot on the cloth. Repeat this process for a total of ten times. 7. Wipe away excess polish from the edges using a Kimwipe. Rinse well with deionized (DI) water. Wipe off excess water using a Kimwipe.
Decreasing the particle size of the polishing paste smooths out the surface and improves the deposition of the PEDOT-phage film in the subsequent steps.
10. Place electrode in a vial containing DI water, and immerse the setup in a water bath sonicator. Sonicate 10 min. Rinse with DI water. Cyclic voltammetry: the three electrode setup 12. Flame-clean the platinum film electrode with a butane torch. Rinse with water. Clamp electrode, and immerse it into the phage-EDOT solution.
A platinum electrode is used as the counter-electrode.
13. Rinse the Ag/AgCl reference electrode. Clamp electrode and immerse it in the phage-EDOT solution.
The Ag/AgCl electrode serves as the reference electrode.
14. Clamp and immerse the Au working electrode into the phage-EDOT solution; place the setup in a Faraday cage.

The electrodes should not be in contact with each other.
15. Connect all electrodes to the potentiostat.
16. Cycle the potential with the following parameters:    22. Connect electrodes to the potentiostat.
23. Acquire five consecutive electro interstitial scanning (EIS) scans: 50 frequency data points spanning 0.1 Hz to 1 MHz with a 10-mV voltage modulation amplitude.
There should be no significant drift in the resistance or impedance values, and the impedance should be monitored to identify drift (see Troubleshooting section and Fig. 10).
This acquisition provides the native resistance (R o ) of the native film, the virus-PEDOT film with no analyte bound.

Wrap the virus-PEDOT films with additional ligands
24. Rinse electrode and incubate in a 1.5 ml-microcentrifuge tube containing 200 μl of K CS -1 at a concentration of 0.5 μg/μl. Gently shake at room temperature for 15 min.
The interaction between K CS -1 and the virus particles is electrostatic. Since the negative charges on the virus surface are required for incorporation into the PEDOT films and wrapping with K CS -1, the virus-PEDOT films were wrapped after the synthesis of the virus-PEDOT films.
The electrode remains clamped during this incubation. The K CS -1 solution is reusable, but a fresh solution should be used for each experiment, as described above in step 16.

Rinse electrode with DI water and then with PBF-Tween.
Care should be taken to wipe off any excess wash liquid adhered to the insulator on the electrode, to avoid any changes in concentration especially for the K CS -1 and PSMA incubation steps.
26. Optional: Check for resistance changes after phage wrapping by acquiring five consecutive EIS scans, each with 50 frequency data points spanning 0.1 Hz to 1 MHz with a 10-mV voltage modulation amplitude.
In our experiments, the resistance of the films did not undergo any significant change upon phage wrapping with K CS -1. However, this possibility should be tested for new peptide wrappers. The dynamic, non-covalent interaction between the K CS -1 wrappers and the viruses in the films could potentially result in decreased levels of K CS -1 wrapped on the phage. However, PSMA binding to the film could reduce the possibility of the K CS -1 wrappers detaching due to its synergistic binding.

PSMA (analyte) binding
27. Incubate the wrapped virus-PEDOT film with 200 μl PSMA in PBF-Tween buffer (of a desired concentration as shown, for example, in Fig. 8) for 30 min with shaking.
Typically, higher concentrations are measured first, but random concentrations can also be used.
28. Rinse electrode, equilibrate and acquire five EIS scans as previously described in steps 20 through 23.
The resistance measurement obtained here is the final resistance of the film (R); this measurement indicates the change in resistance due to PSMA binding.
29. Repeat the above experiment with a different PSMA concentration or an alternative target. Perform appropriate negative controls.
Analyze data 30. Export data from the five scans for both the native film and the film after exposure to PSMA.
Any computing software capable of doing simple calculations and plotting graphs will suffice for the analysis presented here. Microsoft Excel was used for our analysis.
31. Calculate the average resistance and standard deviation values across the five scans corresponding to each frequency for both the films. R = Resistance of the film post-PSMA incubation = R Re R o = Resistance of the native film Each measurement will provide a set of frequencies and their corresponding R Re and R Im values, the real and imaginary components of the impedance, respectively.
32. Calculate the change in resistance caused by PSMA binding across all frequencies as: This value is proportional to the PSMA concentration.
33. Calculate the corresponding error across all frequencies as: where σ i = Standard deviation of the native film σ f = Standard deviation of the film post-PSMA incubation More than one variable is being used to compute the above parameter, and thus, propagation of error is required for the determination of uncertainty.  39. Extrapolate the curve to lower concentrations for computing the theoretical limit of detection, using the parameters obtained, n, K d and Y max .
The experimentally observed LOD is determined as the PSMA concentration for which the R/R o is at least three-fold over background. The same principle was applied to the extrapolated data, and provides the theoretical limit of detection.

REAGENTS AND SOLUTIONS
Use Milli-Q water or equivalent in all recipes and protocol steps.

Azide-and alkyne-functionalized peptides
The lyophilized peptides can be stored indefinitely at −20°C or dissolve the lyophilized peptides in a solution of acetonitrile and HPLC-grade water to a final concentration of 200 μM, with the percentage of acetonitrile as needed for solubility. The solutions can be stored indefinitely at −20°C.

M13 bacteriophage viruses
M13 bacteriophage viruses serve as the scaffold for the receptors in the bioaffinity matrix of the biosensor. The viruses have an ssDNA genome, which is encased in a protein coat comprising ß2700 copies of a single major coat protein (P8), and five copies each of the four minor coat proteins (Welsh et al., 1998;Lowman and Clackson, 2004). The encapsulated DNA can be modified to display peptides or proteins on the surface of the phage as a fusion to the termini of the coat protein. For phage-2, the gene encoding the peptide ligand is fused to the N-terminus of the P8 coat protein. Furthermore, the phage propagation protocol could also be modified to simultaneously display two genetically encoded ligands on phage (Mohan and Weiss, 2014). In each case, the copy number of the displayed peptide ligands on the surface of the phage is much lower in comparison to the total number of P8 coat proteins. Hence, the phage are wrapped with additional chemically synthesized ligands to increase the apparent concentration of ligands in the bioaffinity matrix without increasing the phage concentration. Wrapping phage with additional ligands provides a quick method to increase the affinity and sensitivity of the device due to the enhanced avidity effect.
For the generation of the secondary recognition ligands, the wrappers are synthesized in two parts. The first half consists of an oligolysine (K 14 ) peptide, which wraps around the phage surface due to electrostatic interactions (Lamboy et al., 2008). Carboxylatebearing residues near the N-terminus of the P8 coat protein impart a high negative charge to the phage surface. The K 14 peptide is coupled to pentynoic acid during the synthesis process, providing an alkyne functionality for the click reaction. The second component of the wrapper is the peptide ligand to PSMA. The peptide ligand is similarly synthesized and functionalized with an azide moiety by coupling to 4-azidobutanoic acid. The two components are subsequently linked together by the click reaction.

Phage infectivity
A key step in the phage propagation protocol is the infection of the bacterial culture with the helper phage. The helper phage encodes the proteins necessary for propagation, and the assembly of the virus. The phagemids encode an antibiotic resistance gene different from the helper phage. The addition of both antibiotics to the culture ensures the presence of both phagemid and helper phage DNA in each bacterial cell. For further details of phage propagation and design, we refer the reader to various books on this topic (Kay et al., 1996;Lowman and Clackson, 2004).

Multiplicity of infection
The infection of KO7 phage into the bacterial cell proceeds through the F pili, which are the receptors for phage infection. The number of F pili present per bacterial cell are few and limited (Lowman and Clackson, 2004). Thus, the multiplicity of infection (MOI) ensures high infection efficiency (Equation 1). Additionally, the density of bacterial cell culture at the time of infection is also crucial. Cultures grown past log phase have high cell density, and the expression of pili decreases, which results in lower infection efficiency.

MOI(m) = number of virus particles number of bacterial cells
Equation 1 The number of virus particles actually infecting a cell can be estimated by Poisson distribution as: Figure 7 Polymerization of EDOT in the presence of (A) LiClO 4 or (B) Phage-2, followed by wrapping with K CS -1 (green and blue). Thus, for 99% infection efficiency, the ideal MOI is 4.6.

Biosensing theory
The bioaffinity matrix in the biosensor consists of a composite film of a conductive organic polymer (PEDOT) and viruses deposited on a planar gold electrode. EDOT undergoes electrochemical polymerization in the presence of LiClO 4 to form cationic PEDOT units, which associate with perchlorate ions while depositing onto the electrode ( Fig. 7; Sharma et al., 2012). Polymerization of EDOT in the presence of phage particles leads to the generation of the virus-PEDOT film due to the incorporation of phage particles as the counterions during deposition.
Ligand binding interactions are enhanced due to the cooperative effect of the two ligands present on the surface of the phage, one genetically encoded and the other chemically synthesized. This bidentate binding mode combined with the saturation binding effect can be quantified, and its parameters computed using the Hill equation (Equation 3; Hill, 1910):

Equation 3
Where, The Hill coefficient is a measure of cooperativity present in the binding interaction. A value of 1 indicates no cooperative binding interactions. A value >1, as seen in Figure 8 indicates positive cooperativity, whereas a value <1, indicates negative cooperativity. The bidentate binding mode of ligands 1 and 2 (arising from the combination of phage-2 and K CS -1) results in a Hill coefficient of 1.5, which demonstrates the synergy of the two ligands in cooperatively binding to PSMA.

Optimizing the combination of ligands
The choice of ligands coating the phage surface might require careful selection to optimize the sensitivity of the biosensor. For example, two ligands with negative cooperativity would be poor choices for biosensor development. Fortunately, phage display selections typically result in large numbers of possible ligands. Various scenarios, their causes, and suggestions for improvement are summarized in the Figure 9 flowchart. For non-competing ligands, the choice of the genetically displayed and chemically synthesized ligand could also be a crucial factor, and the orientation of ligands following bioconjugation should be considered.

Phage propagation
Acquiring a phage stock free from contamination is very important for obtaining high sensitivity and specificity for analyte detection. During phage propagation, it is essential to meticulously follow all the steps of the protocol. Possible sources of error and points of concern have been incorporated in the notes for each step in Basic Protocol 2. Close attention should also be paid to the yields obtained for phage-displayed peptide. Unforeseen circumstances such as errors with, for example, MOI calculations could lead to the propagation and packaging of KO7 phage.

The cycloaddition reaction
The choice of azide-and alkynefunctionalized peptides governs the identity of the solvent used for the reaction. If precipitation is observed in the reaction mixture, different temperature conditions (e.g., heating) or solvents could be tried. If the reaction results in low yields, Cu(I)-stabilizing ligands such as TBTA could be used.

Figure 8
R/R o of the film increases with higher concentrations of PSMA.

Figure 9
Flowchart describing the process of designing and planning a ligand combination for the detection of analyte with increased sensitivity. The commonly observed phenomena are listed here alongside possible solutions, including troubleshooting the ligand combination using biological assays such as enzyme-linked immunosorbent assay. The figures provide representative data for each scenario. Phage concentrations are plotted along the x-axis and the assay response along the y-axis.

Biosensing
A common problem encountered with biosensing is drift observed in the impedance values during EIS. The possible causes leading to drifting values and the corresponding solutions are discussed in Figure 10. Non-specific binding observed during the biosensing experiments could be attributed to impurities in the materials or deteriorated reagent stocks. Phage and peptide purity is essential for low background measurements.

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Figure 10
Flowchart listing the possible problems, the corresponding sources of errors, and the recommended troubleshooting steps.

Anticipated Results
Basic Protocols 1 and 2 provide the materials necessary for the generation of the bioaffinity layer. These materials can then be applied to the detection of the cancer biomarker, as described in Basic Protocol 3. Once a clinically relevant limit of detection has been obtained for biosensing with virus-PEDOT films, the assay setup can be used for clinical research with biological fluids from cancer patients.

Time Considerations
Once the purified azide-and alkynefunctionalized peptides have been synthesized and purified, Basic Protocol 1 can be completed in 3 to 4 days. Basic Protocol 2 takes a total of 3 days, including the transformation step. The majority of the effort required for phage propagation occurs on day 3 with the phage precipitation. For Basic Protocol 3, each experiment from electrode cleaning to measuring the final resistance upon PSMA incubation requires ß2.5 hr. With previously prepared, equilibrated and calibrated phage-PEDOT films in device form, five redundant measurements could theoretically be completed in minutes or less.