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Title:
Incorporating Peptide Aptamers into Resistive Pulse Sensing

Paper sections:
Introduction
<p>Capable of single molecule resolution resistive pulse sensors, RPS, are becoming popular within a range of biosensing applications. For example they have been applied to the detection of metabolites, 1 proteins, [2][3][4] DNA sequencing, 5-7 epigenetics 8,9 and cellular vesicles. 10,11 An increasingly common strategy in RPS utilises nanomaterials as carriers of recognition elements to selectively bind to the target. 2,3,[12][13][14][15][16][17] The carrier acts as a support for multiple ligands enhancing the signal and facilitating multiplexed assays. 18,19 To-date DNA aptamers have been a common ligand of choice. These are sequence specific single strand nucleic acids, that bind to target analytes via the formation of a tertiary structure. [20][21][22] Once immobilised onto a carrier, the binding of the analyte can be monitored via two mechanisms. The first is a change in size or shape of the DNA. 17 The second is a change in charge density around the carrier, 1,3 measured through a change in translocation velocity, figure 1, providing quantitative information. 2,3,23  In the absence of convection and electroosmosis, the number of carriers that translocate the sensor, and thus the number of analytes, is related to the size of the carrier and its charge/ zeta potential. Hence most studies within RPS utilise the inherent charge on the phosphate backbone of the DNA to enhance the translocation frequency. The use of peptides themselves as the recognition element is relatively new. 24 Developing this strategy would allow the field of peptide aptamers developed through phage display libraries to be used. Here we present a simple method to include peptide aptamers onto nanocarriers that maintains a high translocation frequency and produces a concentration dependent signal. By grafting a mixed layer of peptides and short nonbinding DNA to the carriers surface we get dual properties. The DNA provides a base charge, enhancing the translocation of the carrier through the pore, whilst the peptide endows the particles with biorecognition. We demonstrate this approach developing a sensor to C-Reactive Protein, CRP, a key biomarker for inflammation and infection that is monitored in the UK as a potential indicator for Sepsis or Myocardial infarction. Binding of the CRP to the peptide creates a protein corona around the particle, shielding the underlying DNA. This results in a decrease in translocation velocity. We illustrate the effects of the changing the length of the DNA:Peptide, which indicates an optimum length of the for the DNA. This simple approach can be easily adapted to other peptides and does not require the peptide itself to undergo any modifications.</p>

Chemicals and Reagents
<p>Unless stated chemicals are purchased from Sigma-Aldrich UK, with no modification or purification. C-Reactive Protein (C4063), Phosphate buffered saline (PBS, P4417) consisted of 0.0027M KCL 0.137 M NACL, 0.01M Phosphate buffer, pH 7.2 and DNA: aptamer 5'-CGAAGGGGATTCGAGGGGTGATTGCGTGCTCCATTTGGTG [BtnTg]-3'. The nonbinding DNA aptamer had the sequence 5'-[T]n [BtnTg]-3', where n is the length. The CRP peptide aptamer sequence was Btn-GGGEWACNDRGFNCQLQR-OH (purchased from Mimotope, Australia at 70% purity, HPLC). The Calibration particles and Nanopores purchased from Izon Science Limited termed. The calibration particles termed CPC200 (mode diameter 210nm, 1x10 12 particles/ml). The nanopore used in this experiment is NP200 with an analysis range of 85-500nm. Superparamagnetic beads, SPB, were purchased from Ademtech (Bio-Adembeads Streptavidin plus 0321) and were functionalised with streptavidin (mode diameter 120-150nm, concentration 1x10 12 particles/ml). Bovine Serum Albumin (BSA) was used as the control protein.</p><p>Assay -The streptavidin functionalised magnetic beads (1x10 10 particles/ml) in PBS, were added to varying concentration of peptide or DNA. The particles and aptamer were mixed together for 30 mins before a magnet was used to extract the particles and wash any excess peptide/ DNA unbound to the particles surface. The functionalised particles were then incubated with the target protein for 1 hour before being analysed via the RPS.</p><p>Analysis -All measurements were done using the qNano instrument (Izon Science Ltd, New Zealand) using the Control Suite Software Version 3.3 for the data capture. Details of the setup can be found elsewhere. 25 Briefly, 75µl of PBS was added to the lower fluid cell. Into the upper fluid cell 40µl of PBS (which contain particles during the assay) was added. The electric field was turned on and a stable baseline was achieved. A particle velocity's through the pore was calculated from the pulse width. 26 Here we use the reciprocal of the value at T0.5 (width of the pulse at 50% peak height) to determine relative particle velocity. These values were then normalised to either calibration particles (CPCs00s) or blank peptide functionalised particles ran on the same day, using the same nanopore and experimental conditions.</p>

Results and Discussion
<p>Within RPS experiments each translocation of an analyte/ carrier through the nanopore produces a pulse, figure 1. The magnitude of the pulse, known as the pulse magnitude, ip, is related to the volume of the carrier, and the width or full width half maximum, FWHM, of the pulse relates to its velocity. 26 In the absence of convection, the velocity of the carrier can be proportional to the surface charge or zeta potential. 26,27 Here the velocity is reported as 1/T0.5 values, 13 which we have previously shown capable of monitoring the length and packing density of DNA around SPB's. 26 As the density of the DNA and/ or its length increases the velocity of the SPB through the pore also increases. Here we first modified SPB with a DNA aptamer 40 bases long that is known to bind to CRP with a Kd of 16.2 nM. 28 As we increase the concentration of aptamer the velocity of the SPBs increases, blue curve figure 2a, up to circa 50 nM, where the velocity remains constant. At this point we have either saturated the surface of the SPB's with DNA or can no longer observe a difference in velocity as the number of DNA's increase. As a comparison the velocities of SPB's modified with a shorter nonbinding DNA only 10 bases in length is also shown, figure 2a (orange curve). Again an increase in velocity is observed as the concentration of DNA is increased, and the velocity remains constant over concentrations of 100 nM. We note that the velocity of the shorter DNA was always slower than the longer aptamer which agrees with previous studies. 26 Next a peptide was immobilised onto the carrier. The peptide has an isoelectric point of 5.8 and resulted in a small increase in velocity, figure 2b. The peptide aptamer used here has been shown to bind CRP with a Kd of 1 nM. 29 As well as a decrease translocation velocity the peptide modification also resulted in a greatly reduced translocation frequency, which we attribute to the lower charge density. The hypothesis was made that the translocation frequency of peptide modified carriers could be increased by grafting both DNA and peptide onto the same particle. Thus the shorter 10 base DNA and peptide were added in equal concentrations which improved the signal, figure 2b "mix". To illustrate the typical response when a protein binds to a DNA aptamer, CRP was incubated with the aptamer modified carriers. Figure 3 shows the change in velocity versus concentration of CRP protein. A small decrease in velocity was observed, which was shown to be consistent over several nanopores, and assays. As a control BSA was added at high concentrations and no change in velocity was observed, figure 3b orange data point. The binding of a protein to a DNA aptamer results in a change in its tertiary structure resulting in a decrease in translocation velocity, figure 3a. 2,30 The magnitude of the velocity change has been shown to be dependent upon the shape of the DNA as well as the relative location of the binding event with respect to the carriers surface. 30 In contrast the binding of the CRP to a peptide only modified SPB results in no observable change in velocity even at high concentrations of target, data not shown. As the protein has a similar ISP to the peptide, 5.2 and 5.8 respectively, we conclude that the formation of a protein corona around the carrier did not sufficiently change the change density and relative velocities. Modifications to peptide sequences to endow them with charge or other functionalities is possible, however any changes to an aptamer sequences risks modifying their properties and affinities to the target. Thus, to exploit existing peptide aptamers within current RPS strategies we opted to modify the nanocarrier rather than the peptide. Drawing on previous work where RPS has been able to monitor protein corona formation. 31 CRP was added to SPBs with a mixed surface (1:1 ratio short DNA and peptide) shown schematically in figure 4a. As the concentration of CRP is increased the velocity of the particle decreases, figure 4b. There is an excellent linear relationship between 0 and 2.5 M CRP which correlates to the clinically relevant levels for monitoring inflammation. At concentrations over 2.5 M the surface of the particles become saturated with CRP protein. The binding of the CRP to the surface results in the formation of a protein corona around the carrier, which shields the charge on the underlying DNA. Here the peptide is circa 6.3 nm in length and the shorter DNA circa 3.5nm assuming a linear structure. The length of the DNA was specifically chosen to be shorter than the peptide so that it did not inhibit the binding. It was interesting to note that the change in velocity for the mixed surface is larger than the DNA aptamer alone shown within figure 3. This simple approach to adapt nanocarriers for peptide aptamers may enhance the field of RPS sensors. Different lengths of DNA were tested to see if the ratio of DNA:peptide length effected the signal. Figure 5 shows the relative velocity of the carriers when the length of the DNA is increased from 5 to 25 (orange). The different lengths of DNA were mixed with equal concentrations of peptide on the carrier's surface (blue). As seen in figure 2 increasing the length of the DNA increases the velocity, and the peptide:DNA mixture is always slower then the DNA only coated particles. The addition of the CRP causes the greatest change in velocity when the DNA is 10 bases in length and future experiments and simulations are planned to explore the optimum ratio between peptide and DNA length and packing density to further enhance the signals.</p>

Conclusions
<p>Here we present a simple strategy to modify nanocarriers with peptide aptamers for the detection of biomarkers. The modification of a nanoparticle with a peptide aptamer to CRP was monitored in a small increase translocation velocity. The binding or CRP to the peptide however could not be monitored. Unlike the binding of CPR to a DNA aptamer, there was an insufficient change in charge density around the nanocarrier to result in a change in translocation velocity. By modifying the surface of the particle with peptide aptamer and a shorter nonbinding DNA oligo, the binding of the target to the peptide creates a corona around the carrier, which shields the phosphate groups of the underlying DNA. This results in a measurable change in electrophoretic mobility of the carrier. The assay had excellent linear response.</p>
The use of nanocarriers within resistive pulse sensing, RPS, aids the detection and quantification of analytes. In the absence of convection, the signal strength and frequency can dependent upon the electrophoretic mobility of the nanocarrier/ analyte. Here we have developed a simple strategy to incorporate peptide aptamers onto RPS assays with enhanced electrophoretic signals. Using a hybrid DNA-Peptide nanocarrier an existing peptide was incorporated into a rapid assay without having to engineer or modify the peptide sequence. The surface of a nanocarrier is coated with a mixture of peptide aptamers and a nonbinding DNA. The binding of the target to the peptide creates an "analyte corona" which shields the phosphate groups of the underlying DNA. This results in a change in electrophoretic mobility of the nanocarrier. The signal is concentration dependent and is illustrated using a peptide to a key biomarker of infection, C-Reactive Protein, CRP. As a comparison we also show the binding of the CRP to a DNA aptamer. This universal approach can be easily adapted to other peptides without the peptide itself to undergo any chemical modifications opening new opportunities and applications in RPS strategies.