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Targeted Enlargement of Aptamer Functionalized Gold Nanoparticles for Quantitative Protein Analysis Feng Li *,† , Jingjing Li, Yanan Tang, Chuan Wang, Xing-Fang Li and X. Chris Le * Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB T6G 2G3, Canada; [email protected] (J.L.); [email protected] (Y.T.); [email protected] (C.W.); [email protected] (X.-F.L.) * Correspondence: [email protected] (F.L.); [email protected] (X.C.L.); Tel.: +1-905-6885550 (ext. 6136) (F.L.); +1-780-4926416 (X.C.L.) † Current Address: Department of Chemistry, Brock University, St. Catharines, ON L2S 3A1, Canada. Academic Editors: Jens R. Coorssen, Alfred L. Yergey and Jacek R. Wisniewski Received: 1 November 2016; Accepted: 18 December 2016; Published: 22 December 2016

Abstract: The ability to selectively amplify the detection signals for targets over interferences is crucial when analyzing proteins in a complicated sample matrix. Here, we describe a targeted enlargement strategy that can amplify the light-scattering signal from aptamer-functionalized gold nanoparticles (Apt-AuNP) with high specificity for quantitative protein analysis. This strategy is achieved by labeling target proteins with competitively protected Apt-AuNP probes and enlarging the probes with gold enhancement. This competitive protection strategy could effectively eliminate nonspecific protein adsorptions from a sample matrix, leading to a highly specific labeling of the target protein. As a result, the subsequent amplification of the light-scattering signal by gold enhancement only occurs in the presence of the target protein. This strategy was successfully demonstrated by analyzing human α-thrombin in human serum samples in a Western blot format. Keywords: aptamer; protein quantification; Western blot; gold enlargement

1. Introduction While the discovery-based proteomic strategies profile protein expressions, interactions, and pathways from a biological sample at global levels, targeted protein or proteome analysis is hypothesis-driven and focuses on a subgroup of proteins of interest [1]. Although strategies that are enabled by mass spectrometry and bioinformatics, such as selected reaction monitoring (SRM) and multiple reaction monitoring (MRM), are revolutionizing the field of targeted protein and proteome research, techniques making use of polyacrylamide gels and immunomembranes, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Two Dimensional (2D)-gel electrophoresis, and Western blotting are still the major workhorses in most biochemical laboratories for the quantitative protein analyses [2]. Therefore, it is worthwhile to develop novel molecular probes that are sensitive and robust for the detection of immunoblotted proteins. Here, we describe a facile signal amplification strategy for immunoblotted proteins via the use of aptamer-functionalized gold nanoparticles (Apt-AuNPs). AuNPs have shown great promise and exciting opportunities for biomolecular analysis due to their unique resonance light-scattering properties and versatility for bioconjugations [3]. Compared to conventionally-used fluorescent or luminescent labels, AuNPs offer high light-scattering intensities, quenching and photo-bleaching resistance, and colorimetric signal read-out that enables robust detection of target molecules [4,5]. These advantages have spurred the development of diverse AuNP-based assays for the detection of nucleic acids [6–8], proteins [9–13], pathogens [14], and cancer cells [15]. However, surfaces of AuNPs are known to cause strong nonspecific binding via electrostatic Proteomes 2017, 5, 1; doi:10.3390/proteomes5010001

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and hydrophobic interactions [16,17]. Consequently, when analyzing targets in complicated sample matrixes, e.g., human serum, any nonspecifically adsorbed AuNP labels may contribute to backgrounds, leading to possible false positive results and compromising detection sensitivity [12,18]. Thus, it is essential to reduce or eliminate nonspecific bindings when applying AuNPs for analyzing targets in complicated sample matrixes. To achieve this goal, we have previously developed a competitive protection strategy that is able to effectively eliminate nonspecific bindings between aptamer functionalized AuNPs (Apt-AuNP) and interference human serum proteins by controlling the DNA assembly on Apt-AuNPs [18]. Here, taking advantage of the competitive protection strategy, we further develop a targeted enlargement method that is able to amplify the light-scattering signal from the protected Apt-AuNP probes by catalytic deposition of metal onto AuNPs for colorimetric analysis of human serum samples in a Western blot format. Catalytic deposition of metal onto AuNPs, e.g., silver enhancement or gold enhancement, has been widely used as signal amplification strategies in scanometric assays and other colorimetric immunoassays [6,9–14]. It has been demonstrated that the light-scattering intensity can be amplified over 5 orders of magnitude by enlarging AuNP labels with silver or gold enhancement [19]. However, the high amplification capacity is also challenged by the strong background raised from nonspecifically bound AuNPs to interferences associated with the target. Here, we reason that the competitive protection strategy can overcome this challenge by effectively eliminating nonspecific adsorptions of Apt-AuNPs to interferences, leading to a targeted enlargement of Apt-AuNPs specifically to the minute amount of target proteins in a complicated sample matrix. 2. Materials and Methods 2.1. Materials Human α-thrombin was purchased from Haematologic Technologies Inc. (Essex Junction, VT, USA). Solution of AuNPs (10 nm in diameter), Bovine serum albumin (BSA), human serum (product number, P2918) and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma (Sigma-Aldrich Co. LLC., Oakville, ON, Canada). The aptamer for human α-thrombin (50 -SH-(CH2)6-GGT TGG TGT GGT TGG-30 , and 50 -FAM-GGT TGG TGT GGT TGG-30 ), and protection DNA PolyA-Oligo10 (50 -AAA AAA AAA AAA AAA AAA AAC CAA CCA CAC-30 ), were synthesized and purified by Integrated DNA Technologies Inc. (IDT, Coralville, IA, USA). LI SilverTM enhancement kit) and GoldTM enhancement kit were purchased from Nanoprobes, Inc. (Yaphank, NY, USA). Reagents for SDS-PAGE gel electrophoresis and Western blot were purchased from BioRad Laboratories (BioRad Laboratories LTD. Mississauga, ON, Canada) and Fisher Scientific (Thermo Fisher Scientific Inc. Mississauga, ON, Canada). The binding buffer contained 100 mM NaCl, 20 mM Tris-HCl, 2 mM MgCl, 5 mM KCl, 1 mM CaCl2, and 0.02% Tween 20. The washing buffer contained 1× phosphate buffer saline (PBS), and 0.1% Tween 20, pH 7.4. The protein loading buffer (10 mL) was made of 800 mg SDS, 4.0 mL glycerol, 0.4 mL 2-mercaptoethanol, 2.0 mL Tris-HCl at pH 6.8, and 8 mg bromophenol blue. 2.2. Preparation and Characterization of Apt-AuNPs and Protected Apt-AuNPs A thiolated aptamer probe was received in a disulfide form. Prior to use, it was activated by 50 µL of 5 mM TCEP in 100 mM Tris-HCl for 1 h at room temperature. This solution was then added to 1 mL AuNPs solution, and the mixture was placed at 4 ◦ C for 16 h. To this mixture was slowly added 100 µL of 2 M NaCl, and followed by 10 s sonication. This process was repeated five times with 20 min intervals to maximize the aptamer loading amounts. The final solution was stored at 4 ◦ C for 24 h. Then, it was centrifuged at 17,000× g for 1 h to separate the AuNPs from the unreacted reagents. The AuNPs were washed three times with 10 mM Tris-HCl (pH 7.4), and then redispersed in 1 mL of 10 mM Tris-HCl (pH 7.4). The aptamer modified AuNPs solution was stored at 4 ◦ C when not in use and was found to be stable for more than two weeks.


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To prepare Proteomes 2016, 4, xthe protected Apt-AuNPs, Apt-AuNPs were treated using the following protocol. 3 of 8 Protection DNA (PolyA-oligo10) was added to Apt-AuNP solution, and the final concentrations of both aptamerand andthe theprotection protection oligo oligo were kept DNA in in both thethe aptamer kept to to 11 μM. µM.The Theaptamer aptamerand andprotection protection DNA mixture wereallowed allowedtotohybridize hybridize for for 1.5 1.5 h h at at room was then diluted thethe mixture were room temperature. temperature.The Thesolution solution was then diluted times with bindingbuffer bufferfor forWestern Westernblot blotapplications. applications. 10 10 times with binding BothApt-AuNPs Apt-AuNPs and and protected protected Apt-AuNPs Apt-AuNPs were Both were characterized characterized by by UV/Vis UV/Visabsorption absorption spectrometry (Figure 1). A Lambda 35 UV/Vis absorption spectrophotometer (PerkinElmer LAS spectrometry (Figure 1). A Lambda 35 UV/Vis absorption spectrophotometer (PerkinElmer LAS Canada INC., Woodbridge, ON, Canada) was used to collect extinction spectra of Apt-AuNPs and Canada INC., Woodbridge, ON, Canada) was used to collect extinction spectra of Apt-AuNPs and protected Apt-AuNPs from 400 nm to 700 nm. protected Apt-AuNPs from 400 nm to 700 nm.

Figure 1. Characterization of gold nanoparticles (AuNPs), aptamer-functionalized gold nanoparticles Figure 1. Characterization of gold nanoparticles (AuNPs), aptamer-functionalized gold (Apt-AuNPs), competitively Apt-AuNPs using a UV/Vis using Spectrophotometer. nanoparticles and (Apt-AuNPs), andprotected competitively protected Apt-AuNPs a UV/Vis The maximum absorbance was observed to be 525 nm for bare AuNPs (black curve) and the Spectrophotometer. The maximum absorbance was observed to be 525 nm for bare AuNPs (black modification of AuNPs with DNA oligonucleotides led to a 4-nm red-shift in the absorbance curve) and the modification of AuNPs with DNA oligonucleotides led to a 4-nm red-shift inspectra the (red and blue spectra curves).(red and blue curves). absorbance

2.3.2.3. GelGel Electrophoresis, Blocking Electrophoresis,Protein ProteinTransferring, Transferring,and and Membrane Membrane Blocking SDS-PAGEseparation separationofofproteins proteins was was performed performed with 12% resolving gel.gel. SDS-PAGE with5% 5%stacking stackinggel geland and 12% resolving were freshly prepared in house. Before loading, protein samples with AllAll thethe gelsgels were freshly prepared in house. Before loading, protein samples werewere mixedmixed with protein proteinbuffer loading on a ratio volume ratio of 3:1 and then heated at for 95 °C for 5 A min. A potential 12 loading onbuffer a volume of 3:1 and then heated at 95 ◦ C 5 min. potential of 12ofV/cm V/cm was applied for gel electrophoresis separation. The proteins on the gel were then transferred was applied for gel electrophoresis separation. The proteins on the gel were then transferred to the to the Polyvinylidene membrane a constant voltage of 120 V for During Polyvinylidene fluoridefluoride (PVDF)(PVDF) membrane with awith constant voltage of 120 V for 1 h.1 h. During this this procedure, the temperature was kept at 4 °C. The blotting buffer (1000 mL) consisted of 200 mLmL procedure, the temperature was kept at 4 ◦ C. The blotting buffer (1000 mL) consisted of 200 methanol, 100 mL 10× Tris-glycine buffer, and 700 mL deionized water. After transferring, the gel methanol, 100 mL 10× Tris-glycine buffer, and 700 mL deionized water. After transferring, the gel was was stained with Coomassie Brilliant Blue R-250 (BioRad Laboratories LTD.) to estimate the stained with Coomassie Brilliant Blue R-250 (BioRad Laboratories LTD.) to estimate the efficiency of efficiency of protein transferring (~60% in our experiment). The membrane was immediately protein transferring (~60% in our experiment). The membrane was immediately blocked with 3% BSA blocked with 3% BSA for 1 h to prevent nonspecific binding. After blocking, the membrane was for 1 h to prevent nonspecific binding. After blocking, the membrane was washed twice with washing washed twice with washing buffer for 10 min each and once with binding buffer. For serum buffer for 10 min each and once with binding buffer. For serum samples, the total protein amount was samples, the total protein amount was determined by measuring absorbance at 280 nm. determined by measuring absorbance at 280 nm. 2.4. Protein Detection by Apt-AuNPs and Silver/Gold Enhancement of Apt-AuNPs 2.4. Protein Detection by Apt-AuNPs and Silver/Gold Enhancement of Apt-AuNPs After gel electrophoresis, transferring and membrane blocking steps, Apt-AuNP probes were Afterand gelincubated electrophoresis, and membrane blocking steps, Apt-AuNP probes were added for 1 h transferring at room temperature. After incubation, the membrane was washed added incubated for 1 h atbuffer room temperature. After incubation, theat membrane was washedThe three threeand times with washing (10 min each time) and dried room temperature. times with washing buffer (10 min each time) and dried at room temperature. The Apt-AuNPs stained Apt-AuNPs stained protein bands on the membrane can be directly visualized by the naked eye. To protein bands the membrane candried be directly visualized by the by naked eye. Toflatbed quantify the protein quantify the on protein amount, the membrane was scanned a desktop scanner (hp amount, driedand membrane wasbyscanned by software a desktop(Adobe flatbed scanner (hp scanjet 4400c) and analyzed scanjetthe 4400c) analyzed imaging Photoshop CS3). For further signal


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by imaging software (Adobe Photoshop CS3). For further signal enhancement, commercial silver or gold enhancement Proteomes 2016, kits 4, x were used, following the recommended protocols for immune-membranes. 4 of 8 enhancement, commercial silver orEnlargement gold enhancement kits were used, following the recommended 2.5. Signal Amplification Using Targeted of Apt-AuNPs protocols for immune-membranes.

For signal amplification using targeted enlargement of Apt-AuNPs, protected Apt-AuNP probes 2.5. to Signal Amplification Using Targetedon Enlargement of Apt-AuNPs were used stain the target protein membrane. After washing three times, the membrane containing the and protected Apt-AuNPs was soaked in goldprotected enhancement solution for Fortarget signalprotein amplification using targeted enlargement of Apt-AuNPs, Apt-AuNP probes three were times used towith staindeionized the targetwater, proteinand on left membrane. Afterdried washing three times, the 5 min, washed to dry. The membrane was imaged by membrane containing the target protein and protected Apt-AuNPs was soaked in gold a desktop flatbed scanner and analyzed by imaging software. enhancement solution for 5 min, washed three times with deionized water, and left to dry. The dried was imaged by a desktop flatbed scanner and analyzed by imaging software. 3. Results andmembrane Discussion Results and Discussion The3.strategy of targeted enlargement of Apt-AuNPs is shown in Scheme 1. To achieve the targeted enlargement,The we strategy construct competitively protected Apt-AuNP probesinby assembling protection of targeted enlargement of Apt-AuNPs is shown Scheme 1. To achieve the DNA targeted enlargement, we construct competitively protected Apt-AuNP probes by assemblingaptamer. (PolyA-Oligo10) onto Apt-AuNPs through a complementary sequence to the conjugated protectiononDNA (PolyA-Oligo10) ontoDNA Apt-AuNPs throughthe a complementary sequence to the to the Once assembled AuNPs, the protection could restrict access of interfering molecules conjugated aptamer. Once assembled on AuNPs, the protection DNA could restrict the access of AuNP surface through a polyA overhang, thereby eliminating nonspecific interactions. In detection interfering molecules to the AuNP surface through a polyA overhang, thereby eliminating of the target protein that is either blotted or captured on a solid substrate, e.g., immunomembrane nonspecific interactions. In detection of the target protein that is either blotted or captured on a or microplate well (in ELISA assay format), or competitive binding of theassay target protein to the aptamer solid substrate, e.g., immunomembrane microplate well (in ELISA format), competitive results inbinding a substitution of the protection DNA, results leading the specificoflabeling of theDNA, target protein with of the target protein to the aptamer in to a substitution the protection leading to theNon-target specific labeling of the target with Apt-AuNPs. Non-target not Apt-AuNPs. molecules couldprotein not displace the protection DNAmolecules because could their binding to displace the protection DNA because their binding to aptamers is much weaker than the aptamers is much weaker than the hybridization between the protection DNA and aptamer sequence. hybridization between the protection DNA and aptamer sequence. After specifically labeling the After specifically labeling the target protein with Apt-AuNPs, a subsequent gold enhancement is target protein with Apt-AuNPs, a subsequent gold enhancement is performed to enlarge the performed to enlarge the labeled Apt-AuNPs. As a enlargement result of theoftargeted enlargement of Apt-AuNPs, labeled Apt-AuNPs. As a result of the targeted Apt-AuNPs, the light-scattering the light-scattering signal can thenenabling be amplified, enabling theofsensitive of the target protein. signal can then be amplified, the sensitive detection the target detection protein.

1. Schematic showing the targeted enlargement of aptamer (blue line) functionalized gold Scheme Scheme 1. Schematic showing the targeted enlargement of aptamer (blue line) functionalized gold nanoparticles (Apt-AuNPs) for immunoblotting-based protein detection. To achieve the targeted nanoparticles (Apt-AuNPs) for immunoblotting-based protein detection. To achieve the targeted enlargement, Apt-AuNPs are protected with protection DNA oligonucleotides (black line is the enlargement, Apt-AuNPs protected DNA (black line is the complementary regionare for the aptamer with strandprotection and green line is theoligonucleotides polyA protection region) before complementary region for the aptamer and green linethe is the polyA protection region) being applied to target proteins. The strand protection DNA restricts access of interfering molecules to before the AuNP surface through The a polyA overhang, thereby eliminating nonspecific interactions. After to the being applied to target proteins. protection DNA restricts the access of interfering molecules AuNP surface through a polyA overhang, thereby eliminating nonspecific interactions. After labeling the target protein with the protected Apt-AuNPs, a subsequent gold enhancement is used to enlarge the Apt-AuNPs that have been specifically captured by the target protein, enabling the colorimetric detection of the target protein.

labeling the target protein with the protected Apt-AuNPs, a subsequent gold enhancement is used to enlarge the Apt-AuNPs that have been specifically captured by the target protein, enabling the colorimetric detection of the target protein. Proteomes 2017, 5, 1

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To demonstrate the feasibility of our strategy, we chose human α-thrombin as a target protein because of its important role in the blood clotting process. A 15-mer DNA aptamer that could bind to theTofibrinogen site the of thrombin used as an affinity ligand. To α-thrombin evaluate theasspecificity of our demonstrate feasibilitywas of our strategy, we chose human a target protein strategy of forits detecting target specifically in process. a complicated sample matrix, we spiked different because important roleprotein in the blood clotting A 15-mer DNA aptamer that could bind concentrations of thrombin into human serum samples, and then separated thrombin from to the fibrinogen site of thrombin was used as an affinity ligand. To evaluate the specificity of our interfering proteins by gel specifically electrophoresis followed bysample the transfer proteins to a strategy for serum detecting target protein in a complicated matrix,ofweallspiked different polyvinylideneoffluoride membrane using electroblotting (Western blot). PVDF concentrations thrombin(PVDF) into human serum samples, and then separated thrombin fromThe interfering membrane was by chosen because of its wide application in Western blot. By separately blotting the serum proteins gel electrophoresis followed by the transfer of all proteins to a polyvinylidene target thrombin interfering serum proteins onto PVDFblot). membranes, we membrane can easily evaluate the fluoride (PVDF) and membrane using electroblotting (Western The PVDF was chosen performance of our strategy by comparing the intensities of different protein bands on the same because of its wide application in Western blot. By separately blotting the target thrombin and membrane. serum proteins onto PVDF membranes, we can easily evaluate the performance of our interfering To select a suitablethe signal amplification strategy for bands naked-eye protein on membrane, strategy by comparing intensities of different protein on the same detection membrane. we first compared the light-scattering signals generated from Apt-AuNPs, Apt-AuNPs with silver To select a suitable signal amplification strategy for naked-eye protein detection on membrane, enhancement, and Apt-AuNPs with gold enhancement 2). Figure 2AApt-AuNPs shows a typical we first compared the light-scattering signals generated(Figure from Apt-AuNPs, with image silver of membrane blotted human α-thrombin (diluted in PBS buffer) after labeling with Apt-AuNPs. enhancement, and Apt-AuNPs with gold enhancement (Figure 2). Figure 2A shows a typical image Themembrane light-scattering intensities from Apt-AuNPs labeled thrombin distinguishable from of blotted human α-thrombin (diluted in PBS buffer) are afterreadily labeling with Apt-AuNPs. the background, resulting in intense red protein bandsthrombin (36 kD) on membrane. The products of The light-scattering intensities from Apt-AuNPs labeled arethe readily distinguishable from the the band sizes and band intensities are proportional to the amount of thrombin in the range of 500 background, resulting in intense red protein bands (36 kD) on the membrane. The products of the band ng to and 5 μg.band The intensities light-scattering signals fromtoApt-AuNP labels were further by depositing sizes are proportional the amount of thrombin in theamplified range of 500 ng to 5 µg. silver or gold onto them through a catalyticlabels reduction silveramplified (Figure 2B) gold ionssilver (Figure 2C). The light-scattering signals from Apt-AuNP wereof further byor depositing or gold By amplifying the signal with silver enhancement, we were able to visually identify as low as 50 ng onto them through a catalytic reduction of silver (Figure 2B) or gold ions (Figure 2C). By amplifying thrombin on the membrane (Figurewe 2B). Gold enhancement further as improved sensitivity the signal with silver enhancement, were able to visually identify low as 50detection ng thrombin on the by forming larger microstructures [9,10], resulting in a detection limit of 5 ng thrombin (Figure 2C). membrane (Figure 2B). Gold enhancement further improved detection sensitivity by forming larger Thus, we chose gold enhancement to amplify the detection signal for Apt-AuNPs in the following microstructures [9,10], resulting in a detection limit of 5 ng thrombin (Figure 2C). Thus, we chose gold studies. enhancement to amplify the detection signal for Apt-AuNPs in the following studies.

Figure 2. Western Western blot blot analysis analysis of of human human α-thrombin α-thrombin in in PBS PBS buffer using Apt-AuNP Apt-AuNP labeling labeling (A), (A), Apt-AuNP labeling followed by silver enhancement (B), and Apt-AuNP labeling followed by gold enhancement (C). Lane 1 contained pre-stained low-range protein standards. Lanes 2–8 contained varying amounts of human α-thrombin: 5 µg (lane 2), 1 µg (lane 3), 500 ng (lane 4), 100 ng (lane 5), 50 ng (lane 6), 10 ng (lane 7), and 5 ng (lane 8). The red arrows indicate α-thrombin.

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enhancement (C). Lane 1 contained pre-stained low-range protein standards. Lanes 2–8 contained varying amounts of human α-thrombin: 5 μg (lane 2), 1 μg (lane 3), 500 ng (lane 4), 100 ng (lane 5), Proteomes 2017, 5, 1 6), 10 ng (lane 7), and 5 ng (lane 8). The red arrows indicate α-thrombin. 6 of 8 50 ng (lane

Having established the signal amplification method for Apt-AuNP labels, we further applied Having established the signal amplification method for Apt-AuNP labels, we further applied this this method to analyze thrombin spiked in human serum. After separating with gel electrophoresis method to analyze thrombin spiked in human serum. After separating with gel electrophoresis and and transferring onto PVDF membranes, target thrombin and interfering serum proteins were transferring onto PVDF membranes, target thrombin and interfering serum proteins were incubated incubated with Apt-AuNPs followed by a subsequent gold enhancement. Figure 3 shows typical with Apt-AuNPs followed by a subsequent gold enhancement. Figure 3 shows typical images of images of protein analyses using Apt-AuNP labeling and gold enhancement on PVDF membranes. protein analyses using Apt-AuNP labeling and gold enhancement on PVDF membranes. In the absence In the absence of the protection DNA (Figure 3A), Apt-AuNP labeling and subsequent gold of the protection DNA (Figure 3A), Apt-AuNP labeling and subsequent gold enhancement was able enhancement was able to amplify the detection signal for human α-thrombin in the diluted human to amplify the detection signal for human α-thrombin in the diluted human serum, indicated by serum, indicated by the protein bands at molecular weight of 36 kD. However, there is a strong the protein bands at molecular weight of 36 kD. However, there is a strong background resulting background resulting from interfering serum proteins. This observation is consistent with the from interfering serum proteins. This observation is consistent with the previous report that high previous report that high concentrations of matrix proteins, e.g., 8% human plasma, could interfere concentrations of matrix proteins, e.g., 8% human plasma, could interfere with the metal enhancement with the metal enhancement of Apt-AuNPs for protein detection [12]. However, by using the of Apt-AuNPs for protein detection [12]. However, by using the competitively protected Apt-AuNPs, competitively protected Apt-AuNPs, we could effectively eliminate nonspecific protein bindings, we could effectively eliminate nonspecific protein bindings, resulting in an amplified scattering resulting in an amplified scattering signal specifically for the target thrombin (Figure 3B). By signal specifically for the target thrombin (Figure 3B). By eliminating the nonspecific bindings from eliminating the nonspecific bindings from interference serum proteins, we could easily identify the interference serum proteins, we could easily identify the target thrombin without the need of any target thrombin without the need of any instrument. To further analyze the data with imaging instrument. To further analyze the data with imaging software, we scanned the membrane with software, we scanned the membrane with a commonly used desktop flatbed scanner. Quantitative a commonly used desktop flatbed scanner. Quantitative information could then be obtained by information could then be obtained by calculating the areas and intensities of protein bands using calculating the areas and intensities of protein bands using Adobe Photoshop software (Figure 4), Adobe Photoshop software (Figure 4), and as low as 30 ng of thrombin was detected in human and as low as 30 ng of thrombin was detected in human serum samples. serum samples.

Figure 3. Western Westernblot blotanalysis analysisofofhuman human α-thrombin a 10-time diluted human serum sample: Figure 3. α-thrombin in in a 10-time diluted human serum sample: (A) (A) protein detection using unprotected Apt-AuNP labelingfollowed followedby bygold goldenhancement; enhancement; (B) (B) protein protein protein detection using unprotected Apt-AuNP labeling detection probe followed Lane 11 detection using using aa PolyA-oligo10 PolyA-oligo10 protected protected Apt-AuNP Apt-AuNP probe followed by by gold gold enhancement. enhancement. Lane contained pre-stained low-range protein standards. Lanes 2–8 contained varying amounts of human contained pre-stained low-range protein standards. Lanes 2–8 contained varying amounts of human α-thrombin: µg (lane (lane 2), 2), 11 μg µg (lane (lane 3), 3), 500 500 ng ng (lane (lane 4), 4), 100 100 ng ng (lane (lane 5), 5), 50 50 ng ng (lane (lane 6), 6), 10 10 ng ng (lane (lane 7), 7), α-thrombin: 55 μg and 5 ng (lane 8). The red arrows indicate α-thrombin. and 5 ng (lane 8). The red arrows indicate α-thrombin.


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Figure 4. 4. Quantitative enlargement of of Apt-AuNPs. Apt-AuNPs. To achieve the the Figure Quantitative protein protein analyses analyses using using targeted targeted enlargement To achieve quantitative information, the PVDF membrane that has been blotted with proteins and undergone quantitative information, the PVDF membrane that has been blotted with proteins and undergone the the targeted-enlargement was scanned a computer a desktop flatbed The scanner. The targeted-enlargement was scanned into ainto computer using ausing desktop flatbed scanner. resulting resulting colored digital image was converted into a grayscale image using Adobe Photoshop. The colored digital image was converted into a grayscale image using Adobe Photoshop. The signal 2 2 ) and signal intensity each protein bandthen was calculated then calculated the product the band intensity of eachofprotein band was as theasproduct of theofband area area (mm(mm ) and the the darkness (0–256). darkness (0–256).

4. Conclusions 4. Conclusions We have have successfully successfully developed developed aa target target enlargement enlargement strategy strategy of of Apt-AuNPs Apt-AuNPs for for colorimetric colorimetric We detection of a minute amount of proteins in human serum samples. The success of developing this detection of a minute amount of proteins in human serum samples. The success of developing this amplification strategy strategy demonstrates demonstrates (1) (1) the the extremely extremely high high selectivity selectivity of of our our competitively competitively protected protected amplification Apt-AuNP probes to target proteins over a complicated sample matrix and interfering proteins; (2) Apt-AuNP probes to target proteins over a complicated sample matrix and interfering proteins; thethe compatibility of of our competitively amplification (2) compatibility our competitivelyprotected protectedApt-AuNP Apt-AuNPprobes probesto to other other signal signal amplification strategies. These features make our strategy ideal to be applied in diverse sensor formats to achieve achieve strategies. These features make our strategy ideal to be applied in diverse sensor formats to targeted signal amplification for protein analyses. targeted signal amplification for protein analyses. Acknowledgments: This work was supported by the Natural Sciences and Engineering Research Council of Acknowledgments: This work was supported by the Natural Sciences and Engineering Research Council of Canada, the Research, thethe Canada Research Chairs Program, Alberta Health, and Canada, the Canadian CanadianInstitutes InstitutesofofHealth Health Research, Canada Research Chairs Program, Alberta Health, Alberta Innovates. and Alberta Innovates. Contributions: F.L., X.-F.L., and X.C.L. conceived and designed Author Contributions: designed the the experiments; experiments; F.L. F.L. J.L. J.L. and and C.W. C.W. performed the paper. performed the the experiments; experiments; F.L. F.L. and andY.T. Y.T.analyzed analyzedthe thedata; data;F.L. F.L.and andY.T. Y.T.wrote wrote the paper. Conflicts Conflicts of of Interest: Interest: The The authors authors declare declare no no conflict conflictof ofinterest. interest.

Abbreviations Abbreviations The following abbreviations are used in this manuscript: The following abbreviations are used in this manuscript: Apt-AuNP Aptamer-functionalized gold nanoparticle SRM Selected Reaction Monitoring Apt-AuNP: Aptamer-functionalized gold nanoparticle MRM Multiple Reaction Monitoring SRM: Selected Reaction Monitoring SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis BSA serum albumin MRM: MultipleBovine Reaction Monitoring TCEP Tris (2-carboxyethyl) hydrochloride SDS-PAGE: Sodium dodecyl sulfate phosphine polyacrylamide gel electrophoresis

BSA: Bovine serum albumin TCEP: Tris (2-carboxyethyl) phosphine hydrochloride References 1.

Boja, E.S.; Rodriguez, H. Mass spectrometry-based targeted quantitative proteomics: Achieving sensitive and reproducible detection of proteins. Proteomics 2012, 12, 1093–1110. [CrossRef] [PubMed] Boja, E.S.; Rodriguez, H. Mass targeted quantitative proteomics: sensitive Aebersold, R.; Burlingame, A.L.;spectrometry-based Bradshaw, R.A. Western blots versus selected reactionAchieving monitoring assays: and reproducible detection proteins. Proteomics 12, 1093–1110. Time to turn the tables? Mol.ofCell Proteom. 2013, 12,2012, 2381–2382. [CrossRef] [PubMed] Aebersold, R.; Burlingame, R.A. Western blots versus selected reaction monitoring Saha, K.; Agasti, S.S.; Kim, C.;A.L.; Li, X.;Bradshaw, Rotello, V.M. Gold nanoparticles in Chemical and Biological Sensing. assays:Rev. Time to turn tables? Mol. Cell Proteom. 2013, 12, 2381–2382. Chem. 2012, 112, the 2739–2779. [CrossRef] [PubMed] Saha, K.; Agasti, S.S.; Kim, C.; Li, X.; Rotello, V.M. Gold nanoparticles in Chemical and Biological Sensing. Chem. Rev. 2012, 112, 2739–2779.

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