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Direct Detection of alpha-1 Antitrypsin in Serum Samples using Surface Plasmon Resonance with a New Aptamer-Antibody Sandwich Assay Suhee Kim, and Hye Jin Lee Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01192 • Publication Date (Web): 12 Jun 2015 Downloaded from http://pubs.acs.org on June 23, 2015

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Direct Detection of alpha-1 Antitrypsin in Serum Samples using Surface Plasmon Resonance with a New Aptamer-Antibody Sandwich Assay Suhee Kim and Hye Jin Lee*

Department of Chemistry and Green-Nano Materials Research Center, Kyungpook National University, 80 Daehakro, Buk-gu, Daegu-city, 702-701, Republic of Korea

*Corresponding author: E-mail address: [email protected]; Tel. + 82 053 950 5336; Fax +82 053 950 6330; Postal address: Department of Chemistry and Green-Nano Materials Research Center, Kyungpook National University, 80 Daehakro, Buk-gu, Daegu-city, 702-701, Republic of Korea

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Abstract

The challenges associated with performing surface plasmon resonance (SPR) based measurements in serum and other biofluids have continued to limit the applicability of this valuable sensing technology for sensitive bioaffinity measurements of proteins in clinically relevant samples. In this paper, a new sandwich assay is introduced for the quantitative SPR analysis of alpha-1 antitrypsin (AAT), which is a recognized biomarker for Alzheimer’s disease. Detection was performed via the specific adsorption of AAT onto a gold chip surface modified with a DNA aptamer. The measurement dynamic range and also sensitivity in serum were improved with the subsequent surface binding of antiAAT. A methodology was established to measure the target protein in serum and albumin solutions with the results correlated with measurements in buffer only. A comparison between SPR and enzyme-linked immunosorbent assay (ELISA) measurements was also made. The detection of AAT in serum at clinically relevant concentrations was demonstrated with target concentrations as low as 10 fM readily achievable.

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Introduction Achieving an early diagnosis or risk assessment of neurodegenerative conditions such as Alzheimer’s disease (AD) via the analysis of easily accessible body fluids is an essential aspect of improving upon the current standards for patient care and treatment. A number of studies of potential protein biomarker candidates in serum, plasma and cerebrospinal fluid samples have been reported to identify changing levels between healthy and AD patients.1-8 Beta amyloid and Tau proteins are the most established neurochemical indicators with other examples including apolipoproteins E (ApoE), apolipoprotein J (ApoJ), factor H, transferrin, and transthyretin.5,8,9 In addition, increasing levels of alpha-1 antitrypsin (AAT) has been associated with neurodegenerative disease5,8,9 along with a range of other inflammatory and non-inflammatory conditions such as pulmonary emphysema, liver and vascular diseases.3 Measurements restricted to only one or two of the protein targets mentioned above are insufficient to provide a valid snapshot of the disease state. However, the ability to accurately and conveniently detect multiple targets simultaneously in serum remains challenging and requires establishing a series of ligands for specific bioaffinity interactions in tandem with robust multiplexed sensing platforms.

In this article, we introduce a new DNA aptamer/antibody sandwich assay pairing for the detection of alpha-1 antitrypsin (AAT) using surface plasmon resonance (SPR). While SPR is now a leading technique for the label-free detection of a wide range of biomolecules either in multi-channel or array formats,10,11 there have been relatively few reports involving direct detection in biological fluid samples12-20 due to the challenges associated with non-specific adsorption. SPR sensing of target proteins in crude serum via specific via direct affinity interactions has been demonstrated,16-20 as well as achieving greater signal enhancement and 3

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selectivity via the use of a secondary probe conjugated to particles.16,21-26 In addition, localized SPR measurements has also been explored for the sensing of antibiotin27 and human epididymis secretory protein28 in serum samples. Thus, after characterising the interactions between AAT with antiAAT and the AAT-sepcific aptamer in buffer, we also demonstrate a methodology for the analysis of AAT in serum samples at concentrations as low as 10 fM with a tuneable dynamic range depending on the antiAAT concentration. A series of SPR measurements extending over a range of target concentrations and serum dilutions were compared. Also, a comparison with conventional enzyme-linked immunosorbent assay (ELISA) measurements was performed to further validate the SPR results.

Experimental Section Extended details on the materials used and protocols related to SPR chip preparation, SPR detection and also ELISA measurements are provided in the Supporting Information. Briefly, all in-situ SPR bioaffinity measurements were performed using a Biacore 3000 located at the National Nanofab Center in Daejeon. Both alpha-1 antitrypsin (AAT) and alpha-1 antitrypsin antibody (antiAAT) were obtained from R&D systems. The 5’-amine modified DNA aptamer specific to AAT (5’-H2N-GGG GCA CGT ACG GGC ATC ATA ACA ACA GGC GTG CCC C-3’)29 was purchased from IDT. In addition, ELISA measurements were performed on a Sunrise-basic microplate absorbance reader (Tecan) using an Antitrypsin Human SimpleStep ELISATM Kit (Abcam) with the exception that the same AAT source as for the SPR measurements were employed rather than the AAT solution provided with the kit. All bioaffinity measurements were either performed in PBS buffer pH 7.4 only, or instead human serum (from human male AB plasma; Sigma-Aldrich) or albumin from human serum or 4

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immunoglobulin G (IgG) that had been diluted in PBS buffer. For all buffer, albumin, IgG and serum based measurements, the AAT solution was flowed over the chip surface for a minimum of 1 hour to ensure a steady-state fractional surface coverage was reached for that particular target concentration. In addition, all error bars reported are calculated from an average of at least three repeat measurements.

Results and Discussion Sandwich Assay Detection Methodology: The preparation of the SPR chip and the combination of antiAAT and DNA aptamer probes used for the detection of AAT target molecules is highlighted in Figure 1. First, the AAT specific DNA aptamer is covalently attached to the MUA modified gold film surface. The coupling of an aptamer sequence to a MUA monolayer has also been previously reported by us for bioaffinity detection in serum samples, where it performed better (less nonspecific adsorption) than an antibody covalently immobilized probe.16 The surface adsorption of AAT onto the aptamer-modified chip surface was then investigated via a series of comparative measurements where AAT was suspended in buffer only as well as spiked in human serum and albumin solutions. Further amplification of the SPR detection signal is then obtained by the binding of antiAAT to a different epitope site on the target molecule to complete the sandwich assay. Also, validation of the SPR results utilizing an ELISA sandwich assay is described later in the article.

The first part of this investigation involved establishing that the aptamer and antiAAT pairing both simultaneously have high affinities for AAT and that they can be applied together in a 5

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sandwich assay format. While the DNA aptamer sequence has been described previously in a patent,29 a value for the aptamer-AAT binding affinity as well as verification of the aptamer/AAT/antiAAT sandwich assay complex used here has not been previously published to our knowledge. A series of real-time SPR measurements were thus performed monitoring AAT specific adsorption to both aptamer and antiAAT functionalized gold surfaces. Figure S1 (supporting information) shows plots of fractional surface coverage as a function of AAT concentration for (a) DNA aptamer and (b) antiAAT chips. Langmuir adsorption coefficient values, Kads, of 4.8(±0.1)×107 M-1 and 8.6(±0.2)×106 M-1 were obtained for the aptamer and antiAAT functionalized surfaces, respectively, indicating both probes to have a high binding affinity for AAT. The fractional surface coverage, , was calculated by dividing the R.U. response at each target concentration, C, by the R.U. for the maximum binding of AAT onto the chip. The subsequent data curve fitted with the Langmuir adsorption isotherm (eq. 1):



(1)



which assumes a simple 1:1 interaction between target and probe with no interactions between adsorbed species.30 The interaction between AAT and antiAAT is in good agreement with a previously reported equilibrium value.31 Both surface probe chemistries resulted in a detection limit in the ~1 nM range. In principle, this level of sensitivity may be adequate for analyzing AAT levels in biological fluids,2 however in order to improve selectivity and attain further signal amplification for measurements in serum, the use of a sandwich assay was required.

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To establish that both the aptamer and aniAAT bind simultaneously to different epitope sites on the AAT target, SPR measurements were performed monitoring antiAAT binding to an aptamer-modified chip surface that had been first exposed to AAT. Based on the Langmuir isotherm plots discussed above, a fixed AAT concentration of 200 nM was selected as this results in a relatively high fractional surface coverage. Following a series of SPR measurements (some of which are shown in Figure 2a), a plot of normalized R.U. versus antiAAT concentration was created. Normalized R.U. values were obtained by subtracting the average R.U. of the non-specific control signals, NC1 to NC3 shown in Table S1, from the average R.U. signal obtained from the AAT detection channel signals. Note that in Figure 2(a) all the SPR response curves for AAT detection are higher than that of all the nonspecific signals from the reference channels and this was a requisite for verifying a successful detection. Also, the AAT binding step was performed by flowing target solution over the chip for a minimum of 1 hour to ensure that a steady state surface coverage was reached before then rinsing and injecting the antiAAT. The data in Figure 2(b), shows that the normalized SPR response steadily increased as the antiAAT concentration was varied from 0.5 to 500 nM. The data in Figure 2(b) could also be successfully fitted to a Langmuir isotherm obtaining an adsorption coefficient value of 3.5(±0.3)×107 M-1. This is comparable to the reverse measurement for AAT binding onto an antiAAT surface described above and provides further supporting evidence for the specific adsorption of antiAAT onto the surface immobilized AAT/aptamer complex. The fitted plot is shown in the supporting information (Figure S1c). A further dataset is also shown in Figure S1(d) where a lower fixed AAT concentration of 10 nM was first exposed to the chip prior to antiAAT.

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Sandwich assay optimization: Further investigation of the sandwich assay was first performed in PBS solution to optimize the injected antiAAT concentration. This was necessary to establish the dependence of the SPR dynamic range on both the target and secondary probe concentrations when applying the sandwich assay.16 Figure 3 compares three examples where the antiAAT concentration used was fixed at (a) 100 nM, (b) 200 nM and (c) 500 nM with the corresponding real-time SPR data shown in Figure S2. In each case, antiAAT was introduced following the exposure of the aptamer-functionalized SPR chip surface to numerous AAT target concentrations in the femtomolar range. It can be seen in both Figures 3(a) and 3(b) that a linear SPR signal response range is obtained between 10 – 100 fM (albeit at different slopes). When the fixed antiAAT concentration is increased to 500 nM in Figure 3(c), a linear range between 1 and 20 fM was observed. This highlights that the amplification level can be tuned to different target concentrations via the secondary probe concentration but the measurement dynamic range is correspondingly changed also.

Prior to discussing the detection of AAT in serum, some preliminary measurements were also performed utilizing mixed aptamer/polyethylene glycol monolayers on the SPR chip to further reduce non-specific adsorption. The use of mixed PEG/probe monolayers has been previously reported by several groups,32,33 including ourselves,21,24 for protein adsorption studies. In this work, a 60:40 MUA:PEG-SH monolayer was assembled onto the gold SPR chip surface. The aptamer was then covalently attached to the chip surface via the MUA end carboxyl group as described earlier.

The resultant data obtained is shown in the supporting

information (Figure S3) where the plot of normalized SPR response versus AAT concentration described in Figure 3b is directly compared with a repeat set of measurements for the 60:40 monolayer. As expected, the slope of the linear SPR response region for the 100% 8

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MUA surface is steeper and about half the dynamic range of the 60:40 mixed monolayer. It is clear that the dilution of the aptamer surface density and resulting loss in sensitivity is a more significant issue regarding performance than the gain associated with a 25% reduction in nonspecific adsorption (Figure S3). Consequently, we chose to utilize the 100% MUA chip for serum-based measurements. Furthermore, the DNA aptamer chip surface was selected as this has a higher binging affinity than the antiAAT and that aptamer functionalized SPR chips have been previously reported to exhibit better resistance to non-specific resistance than antibody-coated surfaces.21,24

Serum sample analysis: To tackle the challenge of using SPR directly in serum we performed a series of measurements at various serum dilutions with comparative measurements also performed using a conventional ELISA sandwich assay and plate reader. It was anticipated that a relatively high AAT concentration (micromolar range) could be present in the serum received2 and thus a combination of serial dilution and controlled AAT spiking measurements were designed to investigate both nonspecific adsorption behavior and the accuracy of the SPR measurements. All serum dilutions were performed with PBS buffer (pH = 7.4).

Figures 4 and 5 show the results from a series of SPR measurements involving no spiking (Figure 4) and with spiking (Figure 5) of AAT. Figure 4(a) shows the change in response associated with the introduction of antiAAT in PBS buffer. Prior to this, repeat aptamer chips had been first exposed to serum diluted in PBS at factors ranging from (i) 106 to (vii) no dilution for a minimum of one hour. The results show an increase in antiAAT adsorption as the serum is more concentrated. This trend could be indicative of different levels of AAT 9

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present on the chip surface at different dilutions. However, an increased level of non-specific antiAAT adsorption associated with serum proteins on the chip surface is also possible. The corresponding R.U. values in Figure 4(b) show that the change in signal varies from 3001500 depending upon the serum dilution compared to a maximum overall signal change of ~250 for AAT measurements in buffer only (Figure 3).

Additional non-specific control (NC) measurements are shown in Figures S4 and S5 comparing measurements where the aptamer probe is replaced with two different noninteracting aptamer sequences (NC4 and NC5). For the measurements in buffer (Figure S4) it can be clearly seen that the signal for the 10 fM AAT detection is greater than the representative curves shown for all the NC1 – NC5 controls described in Table S1. Also, Figure S4 shows that a much smaller increase in SPR signal is observed for the NC4 and NC5 controls upon flowing in antiAAT solution compared to the specific aptamer probe measurement.

In order to further confirm that the SPR signal obtained is predominantly due to the specific adsorption of AAT and antiAAT, a combination of serum dilution and AAT spiking measurements were performed. Figure 5 shows representative SPR data curves monitoring the adsorption of antiAAT for (a) 103 and (b) 104 times diluted serum at spiked AAT concentrations ranging from 10 to 100 fM. A series of R.U. plots calculated for a larger range of serum dilutions and spiked AAT concentrations are summarized in Figure 5(c). Interestingly, a linear response was obtained in each measurement series with the R.U. values increasingly offset with respect to the buffer only signal as the serum was less diluted. The slopes of each plot were comparable (ranging from 1.60 to 2.46 R.U./fM) to that of the buffer 10

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only plot (1.67 R.U./fM). This provides strong evidence that the fM range established for AAT detection is reliable and that antiAAT is specifically adsorbing on the chip surface and the SPR signal offset is due to non-specific adsorption but this does not interfere with the formation of the aptamer/AAT/antiAAT complex formation. Furthermore, the dataset in Figure 5(c) allowed us to estimate the original concentration of AAT already present in the serum prior to spiking. This was calculated to be ~35 M (see supporting info) which is comparable with literature values.2 Note that measurements at 100x dilution and lower were performed; however the SPR response was very similar to non-diluted serum for both AAT spiked and non-spiked measurements. This is attributed to a combination of the relatively high AAT concentration originally present in the serum along with increased non-specific adsorption both contributing to a large background SPR response. Consequently, no data series below 1000x dilution are presented in Figure 5.

Further comparison measurements were also performed in human serum albumin solutions of known concentration since this is the most abundant protein in blood plasma with typical levels in the range of 700 M.34 A series of SPR measurements over the same spiked AAT concentration range were then obtained at three different albumin concentrations of 70 nM, 700 nM and 700 M corresponding to serum dilution factors of 10000, 1000 and 0 respectively. Representative SPR sensorgrams are shown in Figure S6, where the signals at all AAT concentrations were at least twice greater than the albumin only response. A clear difference between these results in Figure 5(d) and that shown in Figure 5(c) is that there is a much lower y-axis offset between each data plot at different albumin concentrations compared to the measurements performed in buffer only. This strongly suggests that the nonspecific adsorption of albumin onto the chip surface and also between antiAAT and any 11

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adsorbed albumin is not a significant issue. Instead, the increasing offset observed for the serum-based plots in Figure 5(c) must be affiliated with less common proteins present in the serum. The same trends were also observed for the direct detect of AAT with no secondary antiAAT signal amplification with a systematic signal offset obtained as the serum was diluted while measuring spiked AAT concentrations in the nanomolar range, and from which a native AAT concentration of ~25 M was also separately determined (see Figure S7 and description in supporting information). A further investigation of the nonspecific signal from serum was also performed using IgG (see Figure S8). Similar to the albumin case, there is a relatively small contribution to the background signal observed. This highlights that there is a need to systematically explore in depth other major serum components (e.g. IgA, IgM, transferrin, lipoproteins)35 to understand non-specific adsorption on aptamer functionalized surfaces and this is the subject of a future study.

ELISA measurements: To verify the SPR measurements above and also provide a separate analysis of AAT levels in non-spiked serum, a commercially available ELISA kit was employed. As highlighted in Figure 6(a), a sandwich assay format was selected (rather than a competitive assay) with both buffer calibration and serum measurements performed according to the suppliers protocols. However, it is important to point out here that the AAT used for all SPR and ELISA measurements was a lyophilized form from the same source (R&D systems) as the AAT solution provided with the ELISA kit (Abcam) could not be successfully used when attempting SPR measurements and a direct comparison of ELISA data using both AAT sources resulted in approximately a two-fold difference in signals when comparing at the same concentration. Colorimetry measurements using a microwell plate reader following the HRP catalyzed reduction of TMB and subsequent acidification are 12

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shown in Figure 6 for both serum and albumin solution based measurements. As for the case of SPR, a linear response was achieved in each measurement series with the OD 450 values increasingly offset with respect to the buffer only signal as the serum was less diluted. The slopes of each plot were comparable (ranging from 0.06 to 0.08 OD/nM) to that of the buffer only plot (0.06 OD/nM) obtained at AAT concentrations ranging from 1 to 30 nM. The linear response range is about six orders of magnitude lower than the 10-100 fM range established for the optimized SPR measurements.

Significant differences between the serum and albumin dilution measurements can be seen when comparing Figures 6(b) and 6(c). Repeat measurements at albumin concentrations ranging from 70 nM to 700 M all closely match the buffer only data at different AAT concentrations, which is in contrast to the serum measurements where the linear data plots are increasingly offset with respect to the buffer measurement at different serum dilutions. This result further emphasizes that it is not albumin proteins that are responsible for non-specific adsorption of target and reporter molecules onto the bottom of each microwell. In addition, it can be seen in Figure 6(b) that there is also reasonable overlap for repeat measurements at serum dilution factors of 10,000 and higher. This is expected, as the recommended serum dilution of the ELISA kit manufacturer is between 10,000 and 640,000.

Finally, based on the calibration plots in Figure 6 it is possible to estimate the amount of native AAT in the serum supplied. Based on OD values obtained for non-spiked serum a native AAT concentration of 36 μM was obtained (more details in the supporting information). This agrees with the estimated values stated above from the SPR analysis utilizing both direct and sandwich assay based detection and micromolar concentrations are 13

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typically reported in the literature.2 To obtain an accurate AAT estimation via either SPR or ELISA the appropriate dilution factor has to be established as both techniques are restricted to a particular linear dynamic response range and at lower or higher dilutions the signal response is either saturated or too close to the background controls. This study also highlights the role of both spiked serum and buffer calibration measurements to understand the signal offset associated with serum background contributions and that SPR is an equally valid technique as ELISA for serum diagnostics but can also achieve much better sensitivities.

Conclusions In this article, the application for a new DNA aptamer/antibody sandwich assay pairing specific to AAT protein biomarker has been introduced and successfully applied to measure femtomolar target concentrations in human serum. An extensive series of measurements across a wide range of serum dilutions and spiked target concentrations were performed to further support our results. Also, additional measurements in albumin solutions highlighted that it is the lesser abundant serum proteins that have a much more prominent role in the nonspecific adsorption behaviour observed on the aptamer functionalized SPR chip. Also, the advantages of aptamers over antibodies for preparing biosensing surfaces (e.g. control over attachment chemistries, robustness, etc) are becoming widely recognized and in our experience19,21 we have found aptamer modified SPR chips to outperform antibody-coated surfaces for serum-based analyses. However, the albumin and IgG comparison measurements highlight that further work is required to better understand which serum components contribute the greatest to the non-specific background signal on aptamer-coated surfaces. Certainly, as the availability of new aptamers for different serum biomarkers continues to 14

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improve we anticipate that SPR will become an increasingly valuable technique for multiplexed detection in complex biological matrixes.

Acknowledgement This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2013R1A2A2A03068833).

Supporting Information Further experimental details include materials used, fabrication of aptamer and antibody biochips as well as SPR and ELISA measurements. Figures S1-8 and Table S1 are also included. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Cohen, F. E.; Kelly, J. W. Nature 2003, 426, 905-909. (2) Sun, Y.; Minthon, L.; Wallmark, A.; Warkentin, S.; Blennow, K.; Janciauskiene, S. Derment. Geriatr. Cogn. Disord. 2003, 16, 136-144. (3) Lisowska-Myjak, B. Clin. Chim. Acta 2005, 352, 1-13. (4) Maes, O. C.; Kravitz, S.; Mawal, Y.; Su, H.; Liberman, A.; Mehindate, K.; Berlin, D.; Sahlas, D. J.; Chertkow, H. M.; Bergman, H.; Melmed, C.; Schipper, H. M. Neurobiol. Dis. 2006, 24, 89-100. (5) Liao, P.; Yu, L.; Kuo, C.; Lin, C.; Kuo, Y. Proteomics Clin. Appl. 2007, 1, 506-512. 15

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(6) Rachelefsky, G.; Hogarth, D. K. J. Allergy Clin. Immunol. 2008, 121, 833-838. (7) Sihlbom, C.; Davidsson, P.; Sjogren, M.; Wahlund, L.; Nilsson, C. L. Neurochem. Res. 2008, 33, 1332-1340. (8) Lista, S.; Faltraco, F.; Prvulvic, D.; Hampel, H. Prog. Neurobiol. 2013, 101-102, 1-17. (9) Song, F.; Poljak, A.; Smythe, G. A.; Sachdev, P. Brain Res. Rev. 2009, 61, 69-80. (10) Abbas, A.; Linman, M. J.; Cheng, Q. Anal. Chem. 2011, 83, 3147-3152. (11) Lee, H. J.; Wark, A. W.; Corn, R. M. Analyst 2008, 133, 965-1112. (12) Zeng, S.; Yong, K.; Roy, I.; Dinh, X.; Yu, X.; Luan, F. Plasmonics 2011, 6, 491-506. (13) Jans, H.; Huo, Q. Chem. Soc. Rev. 2012, 41, 2849-2866. (14) Zeng, S.; Baillargeat, D.; Ho, H.; Yong, K. Chem. Soc. Rev. 2014, 43, 3426-3452. (15) Bolduc, O. R.; Masson, J. Anal. Chem. 2011, 83, 8057-8062. (16) Jang, H. R.; Wark, A. W.; Baek, S. H.; Chung, B. H.; Lee, H. J. Anal. Chem. 2014, 86, 814-819. (17) Krishnan, S.; Mani, V.; Wasalathanthri, D.; Kumar, C. V.; Rusling, J. F. Angew. Chem. Int. Ed. 2011, 50, 1175-1178. (18) Masson, J.; Battaglia, T. M.; Khairallah, P.; Beaudoin, S.; Booksh, K. S. Anal. Chem. 2007, 79, 612-619. (19) Phillips, K. S.; Han, J. H.; Cheng, Q. Anal. Chem. 2007, 79, 899-907. (20) Bolduc, O. R.; Masson, J. Langmuir 2008, 24, 12085-12091. (21) Kim, S.; Lee, J.; Lee, S. J.; Lee, H. J. Talanta 2010, 81, 1755-1759. (22) Sim, H. R.; Wark, A. W.; Lee, H. J. Analyst 2010, 135, 2528-2532. (23) Kim, E. J.; Chung, B. H.; Lee, H. J. Anal. Chem. 2012, 84, 10091-10096. (24) Kwon, M. J.; Lee, J.; Wark, A. W.; Lee, H. J. Anal. Chem. 2012, 84, 1702-1707. (25) Spinger, T.; Homola, J. Anal. Bioanal. Chem. 2012, 404, 2869-2875. (26) Baek, S. H.; Wark, A. W.; Lee, H. J. Anal. Chem. 2014, 86, 9824-9829. (27) Yamamichi, J.; Ojima, T.; Iida, M.; Yurugi, K.; Imamura, T.; Ashihara, E.; Kimura, S.; Maekawa, T. Anal. Bioanal. Chem. 2014, 406, 4527-4533. (28) Canaveras, F.; Madueno, R.; Sevilla, J. M.; Blazquez, M.; Plineda, T. J. Phys. Chem. C 2012, 116, 10430-10437. (29) Greving, M.; Woodbury, N. Non-random aptamer libraries and methods for making 2007, WO2007109067 (A2). (30) Wegner, G. J.; Wark, A. W.; Lee, H. J.; Codner, E.; Saeki, T.; Fang, S.; Corn, R. M. Anal. 16

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Figures

Figure 1. Schematic overview of strategy for the SPR detection of AAT. Aptamer probe functionalization of gold SPR chips via the formation of a MUA monolayer followed by covalent cross-linking to an AAT specific DNA aptamer. AAT binding measurements were performed in both buffer and serum with a subsequent anti-AAT binding step for further signal amplification.

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Figure 2. Real-time SPR responses for antiAAT adsorption onto a surface bound AAT/aptamer complex, which was formed in each measurement by first exposing the aptamer surface to a fixed AAT concentration of 200 nM. The subsequent concentrations of antiAAT injected were (i) 10 nM, (ii) 50 nM, (iii) 100 nM and (iv) 200 nM. NC3 is a negative control experiment where 0.5 nM antiBNP was used instead of antiAAT. (b) Corresponding plot of normalized change in SPR response obtained from SPR curves at various antiAAT concentrations.

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Figure 3. Monitoring changes in SPR response (normalized with respect to the average signal of NC1-3 controls) where the antiAAT concentration was fixed at (a) 100 nM, (b) 200 nM and (c) 500 nM for a series of measurements where the SPR chip had already been exposed to different AAT target concentrations. The linear response range in each data series is highlighted by the dotted line.

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Figure 4. (a) Representative real-time SPR responses for the detection of AAT in different dilutions of serum samples. (i) 1,000,000, (ii) 100,000, (iii) 10,000, (iv) 1,000, (v) 100 and (vi) 10 times diluted serum samples. (vii) undiluted serum. 200 nM antiAAT was used. (b) Plot of R.U. signal versus the –log value of the serum sample dilution factor.

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Figure 5.

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SPR measurements of AAT spiked in human serum (a-c) and albumin (d)

solutions. Representative real-time SPR curves are shown where serum was first diluted (a) 1000-fold and (b) 10000-fold prior to AAT addition. In (c), R.U. signals are compared over the same AAT concentration range in (i) buffer only, and at serum dilutions of (ii) 109, (iii) 106, (iv) 105, (v) 104 (vi) 5,000 (vii) 1,000 times. (d) Plots of SPR signals where measurements at different AAT concentrations in (i) buffer only are compared to AAT added to different concentrations of albumin: (ii) 70 nM, (iii) 700 nM and (iv) 700 μM. In all measurements, the AAT concentration was varied from 10 fM to 100 fM, while the subsequent antiAAT concentration injected was fixed at 200 nM.

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Figure 6. (a) Schematic outlining the ELISA-based colorimetric detection of AAT in either serum (S) and buffer. (b) Changes in optical density (OD) at 450 nm measured at a range of AAT concentrations in buffer only and serum solutions diluted by factors of 50,000, 25,000, 10,000, 5,000 and 1,000 prior to spiking with AAT concentrations ranging from 1 to 20 nM. (c) Similar measurements were performed with AAT instead spiked (at concentrations from 1 nM to 20 nM) into albumin solutions with concentrations of 70 nM, 700 nM and 700 μM.

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