Nanobody-Based Electrochemical Immunoassay ... - ACS Publications

Subscriber access provided by UNIV OF CAMBRIDGE

Article

Nanobody-Based Electrochemical Immunoassay for Ultrasensitive Determination of Apolipoprotein-A1 Using Silver Nanoparticles Loaded Nano Hydroxyapatite as Label Huan Wang, Guanghui Li, Yihe Zhang, Min Zhu, Hongmin Ma, Bin Du, Qin Wei, and Yakun Wan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04063 • Publication Date (Web): 02 Nov 2015 Downloaded from http://pubs.acs.org on November 2, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Nanobody-Based Electrochemical Immunoassay for Ultrasensitive Determination of Apolipoprotein-A1 Using Silver Nanoparticles Loaded Nano Hydroxyapatite as Label

Huan Wang,1,† Guanghui Li,1,‡ Yihe Zhang,† Min Zhu,‡ Hongmin Ma,† Bin Du,† Qin Wei*,† and Yakun Wan*,†,‡

†

Key Laboratory of Chemical Sensing and Analysis in Universities of Shandong,

School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, P.R. China ‡

CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai, 201203, P.R. China * Corresponding-Author E-mail: [email protected] (Q. Wei), [email protected] (Y. Wan). 1

These authors contributed equally.

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Nanobodies (Nbs), derived from camelid heavy-chain antibodies, have distinct advantages over conventional antibodies in immunoassay. In this work, Nbs (Nb11 and Nb19) that can bind to different epitopes on apolipoprotein-A1 (Apo-A1) were screened out from an immunized Bactrian camel for the first time. Nb11 were used as capture antibody and fixed on gold nanoparticles (Au NPs) modified screen-printed carbon electrode (SPCE). The silver nanoparticles loaded nano hydroxyapatite (Ag-nHAP) was used as signal tag to label secondary antibody Nb19. A sandwich-type immunological reaction occurred between Apo-A1 and the two Nbs, which brought the Ag-nHAP to the SPCE surface. After the Ag-nHAP were acidically dissolved in the micro electrolytic cell of the SPCE, stripping voltammetric measurement for the released silver ions was performed to obtain an amplified signal. The peak current values increased by the logarithmic values of Apo-A1 concentrations from 10-4 to 50 ng mL-1 under optimal conditions. The detection limit was calculated to be 0.02 pg mL-1. This method was used for the serum samples analysis and achieved satisfactory results. The low cost and high sensitivity make the electrochemical immunosensor suitable for the Apo-A1 detection, which may find promising application in other fields.

2


Page 2 of 25

Page 3 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Apolipoprotein-A1 (Apo-A1) is the primary scaffolding protein of high-density lipoprotein. Since the level of high-density lipoprotein cholesterol is associated with the risk of cardiovascular disease (CVD)1-3, it is vital to be able to measure the level of HDL cholesterol to assess the functionality and patient risk in CVD. However, study showed that high-density lipoprotein cholesterol level alone failed to demonstrate the effects of morbidity and mortality among CVD patients.4 As a result, Apo-A1 was taken into account to evaluate the risk. To our knowledge, only a fast semi-quantitative LC-MS method was reported for Apo-A1 determination.5 There is an urgent need to develop reliable, convenient and accurate diagnostic methods for the detection of Apo-A1. Electrochemical immunoassay for biomarkers has gained increasing interest in clinical examination due to the high sensitivity and operational simplicity.6,7 The specific immunoreaction on the electrode surface between antibody and antigen affords high selectivity for the analyte. However, the instability of conventional antibody demands a careful and rigorous fabrication process. Recently, nanobodies (Nbs), namely variable domains of heavy chain antibodies (VHHs), have aroused considerable interest in the development of electrochemical immunosensors.8,9 Compared with the widely used conventional monoclonal antibodies, Nbs have smaller sizes and higher binding specificity. Nbs also show improved solubility and good thermal and chemical stability, which contribute to the stability of the immunosensor. However, research on Nbs are still less, and most of the research are committed to the drug development. In the present work, a pair of Nbs that can bind 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

to different epitopes on Apo-A1 were screened out from Bactrian camels for the first time. The Nbs (Nb11 and Nb19) can form a sandwich-type immunocomplex with Apo-A1 and were used to fabricate an electrochemical immunosensor for determination of Apo-A1. Many signal amplification strategies based on the increased loading of signal tags have been used for the improvement the sensitivity. Various materials including colloidal gold nanoparticles (Au NPs),10 magnetic nanoparticles,11 mesoporous silicon nanoparticles,12-15 carbon nanotubes16,17 and polymers18,19 have been used to improve the sensitivity by means of loading abundant enzymes,20-23 dyes24-26 or quantum dots27,28. Metal ions show unique electrochemical activity and stripping voltammetric sensors for heavy metal ions have been well exploited.29-31 However, the use of metal ions as signal tags in the fabrication of electrochemical immunosensor were less reported. In our previous report, a novel immunosensor was developed using Fe3O4 to load Pb2+ or Cd2+ as labels.32 However, further improvement of the sensitivity was limited to the adsorption capacity of Pb2+ or Cd2+. Furthermore, new sensing systems based on silver ions deposition were reported.6,33 Adsorption of silver ions was not required and therefore this system was more robust. Another reported strategy involves the conversion of PbS quantum dots (QDs) into Pb2+ ions.34,35 These methods have well demonstrated the prospect of using metal ions as signal tags, but a simple and universal strategy avoiding the complex detection procedure is still urgently needed in this filed. 4


Page 4 of 25

Page 5 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Herein, a nanobody-based electrochemical immunoassay was proposed for sensing of an important protein Apo-A1 with gold nanoparticles (Au NPs) modified screen-printed carbon electrode (SPCE). Au NPs have large surface area for loading Nb11 and amplify signals by accelerating electrochemical signals of silver ions stripping. Hydroxyapatite (HAP) is a biocompatible, nontoxic matrix suitable for loading silver nanoparticles via an ion exchange mechanism with calcium ions. Compared to physical adsorption, ion exchange can trap more silver ions and avoid desorption. The resulting silver nanoparticle loaded nano-hydroxyapatite (Ag-nHAP) serves as a good carrier for labeling Nb19. The sandwich-type immune reaction for Apo-A1 brings the trapped silver nanoparticles onto the micro-electrolytic cell of SPCE, wherein silver ions are thorough released in situ, generating a large stripping voltammetric signal. The highlights of this work lie in: 1) the first explored Nbs for Apo-A1 guarantee the selectivity and stability of the immunosensor. 2) nHAP show excellent ion-exchange capacity with silver ions and biocompatibility with biomolecules. 3) The acid soluble property of nHAP enables the facile release of silver ions. 4) The SPCE provides a micro electrolytic cell, which enables the in-situ detection and enlarges the detection signal due to the trace electrolyte. The proposed immunosensor shows good analytical performance towards Apo-A1 in human serum samples and may find broad applications. ■ EXPERIMENTAL SECTION

Bactrian Camel Immunization and Anti-Apo-A1 Screening. Scheme 1 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

illustrates the workflow of selecting anti-Apo-A1 Nbs. A healthy Bactrian camel was first injected with Apo-A1 antigen with high purity and immunogenicity. After immunization for six times, its blood was collected followed by RNA extraction and reverse transcription to cDNA. After anti-Apo-A1 library construction, library panning, and expression and purification of soluble Nbs, four classes of Nbs were screened and defined as Nb11, Nb19, Nb45 and Nb79. Nanobody Match Pair Assay. Four Nbs, which acted as capture antibodies, were diluted to 3 µg mL-1 with 100 mM NaHCO3 coating buffer (pH 8.2) and coated onto microtiter plates at 4 ºC overnight. Meanwhile, these Nbs were modified with horseradish peroxidase (HRP), which served as detection antibodies. The coating buffer was also used as a control. Next, the four Nbs were conjugated to HRP individually36. The coated wells were washed with phosphate buffer saline (PBS) with 0.05% Tween-20 (PBST) and then added 100 µL antigen (dissolved in 1×PBS with concentration of 5 µg mL-1) at room temperature for two hours’ incubation. After washing the unconjugated antigen with PBST, detection antibodies were added and incubated for 1h. Then 100 µL of 3, 3’, 5, 5’-tetramethylbenzidine (TMB) color liquid was added. The resulting diimine generated by HRP and TMB reaction causes the solution to blue color. At last, 50 µL of 2 M sulphuric acid was added to halt the reaction and the TMB turned yellow. The color was read at 450 nm. Preparation of Nanobody-based Electrochemical Immunoassay. Scheme 2 shows the fabrication and working principle of the immunosensor. Firstly, 2 mg of nHAP with the size of 60 nm were dispersed into 4 mL ultrapure water under 6


Page 6 of 25

Page 7 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ultrasonic wave. Subsequently, 1 mL of AgNO3 solution (10 mM) were added under magnetic stirring and kept for 4 h. The solution color turned from white to gray. The final gray precipitate (Ag-nHAP) was obtained by centrifuging, rinsing with ultrapure water and drying in vacuum. Subsequently, 2 mg of Ag-nHAP was re-dispersed in PBS (4 mL, pH=7.4) and incubated with Nb19 solution (0.5 mL, 10 µg mL-1) for 2 h, followed by centrifugation and rinsing with PBS. The synthesized Nb19/Ag-nHAP was re-dispersed in 2 mL PBS and then incubated with BSA solution (1 mL, 100 µg mL-1) for 1 h. After centrifugation and rinsing with PBS, the Nb19/Ag-nHAP conjugate was re-dispersed in 4 mL PBS and 0.5 mg mL-1 Nb19/Ag-nHAP were finally obtained. For the fabrication of the immunosensor, 20 µL of Au NPs solution was dropped on a SPCE, dried in air and rinsed with PBS. The Au NPs modified SPCE was incubated with Nb11 (10 µL, 10 µg mL-1) for 1 h, and then rinsed with PBS. Subsequently, it was incubated with BSA solution (5 µL, 100 µg mL-1) for 30 min. After rinsing, the SPCE was incubated with Apo-A1 solution for another 1 h. Finally, the Nb19/Ag-nHAP solution prepared in the previous step was dropped onto the SPCE and incubated for another hour. After the final rinsing with PBS, the SPCE immunosensor was ready for use. Electrochemical Measurements. All measurements were performed on the SPCE and 50 µL of acetate buffer solution were used as the electrolyte. HCl solution (10 µL, 10% (V/V)) was added to dissolve Ag-nHAP and release Ag+ for the subsequent stripping voltammetric measurement. LSSV methods were performed 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

from -0.2 to 0.4 V right after the addition of HCl solution with a deposition potential of -0.2 V. EIS was carried out in 50 µL of K3Fe(CN)6 / K4Fe(CN)6 (2.5 mM) and KCl (0.1 M) solution and the frequency was set at 0.1 Hz-100 kHz. ■ RESULTS AND DISCUSSION

Nbs Generation, Purification and SPRi Binding Assay. The details of camel immunization, anti-Apo-A1 phage library construction, library screening and Nbs identification were demonstrated in Figure S1. For expression, the selected Nb plasmids were transformed into WK6, which is a non-suppressor E. coli strain expressing the amber stop codon between VHH and gene III. Therefore, the cells can express soluble Nbs without pIII protein. With the C-terminally hexahistidine (6×His)-tag，the obtained Nbs were purified with Ni−NTA affinity columns. As shown in Figure S2, gel electrophoresis was used to detect the sizes of the Nbs and about 15 kD were obtained, which was consistent with the theoretical value. The concentration of the Nbs was determined by Bradford method, and high purities (>90%) were obtained (Figure S2). Properties such as molecular mass, isoelectric point and yield of the four Nbs were also identification, which was showed in Table S1, the yields of Nbs all in the range from 9.1 to 10.1 mg L−1. When Nb11 was immobilized onto the plate and Nb19 was utilized for photochromic reaction, the experiment presented positive results, and vice versa. However, other combination of Nbs did not showed positive results (Table S2). If two Nbs bind to the same epitope of one antigen, the first Nb which can recognize and get 8


Page 8 of 25

Page 9 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


access to the binding site will make the other Nb unable to be attached to antigen. Therefore, Nb11 and Nb19 can bind to two different epitopes on Apo-A1 were validated. Therefore, Nb11 and Nb19 were chosen to construct the immunoassay and perform SPRi binding assay to measure the affinities to antigen Apo-A1. As shown in Figure 1A and Table S3, Nb11 and Nb19 exhibit high affinity for Apo-A1. The strong binding of Nb11 and Nb19 to Apo-A1 forms the foundation of the new immunoassay. Thermal Stability. The two isolated Nbs (Nb11 and Nb19) were incubated in a thermostatted water bath at 37 ºC for 0.5, 1, and 2 h, and at 70 ºC and 90 ºC for the same durations. The relative activities of Nbs before incubation were regarded as 100%. After thermal treatment, the samples were tested by ELISA to evaluate their activities of direct binding to Apo-A1 coated on a microtiter plate. As shown in Figure 1B, both Nbs maintained nearly 100% activity after incubation at 37 ºC up to 2 h. Nb11 and Nb19 also showed around 100% activity even after incubation at 70 ºC for 2 h. The relative activities of both Nb11 and Nb19 remained above 80%, although slightly decreasing with increasing temperature and increasing time at 90 ºC. The results above showed good thermal stability of the two Nbs. Optimization of Electrochemical Response. Ag-nHAP/Nb conjugate was directly dropped onto the SPCE surface and dried afterwards. LSSV experiments were performed after a deposition time of 150 s. As shown in Figure 2A, approximately 7-fold amplification of electric current was achieved after dissolution of the conjugate with 10 µL of 10% (V/V) HCl solution. This suggests that a large amount of silver ions was released from the Ag-nHAP after acid dissolution, which greatly increased 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the detection sensitivity. Experimental conditions were optimized to maximize the peak current. Figure 2B shows the peak current vs the volume of HCl solution at a deposition time of 60 s. The peak current increased by the HCl volume prior to 10 µL and then reached a plateau at higher volumes possibly due to the full release of the silver ions. Optimization of deposition time is shown in Figure 2C. At a fixed volume of 10 µL HCl solution, the peak current reached a plateau after the deposition time of 180 s, therefore, 180 s was selected as the optimal deposition time. At a fixed volume of 10 µL HCl solution and a fixed deposition time of 180 s, as shown in Figure 2D, 20 µL of Au NPs solution was selected as the optimum volume. The substantial drop as the volume reached 25 µL may be caused by the stacking of Au NPs resulting in the decrease of electrical conductivity. Confirmation of Immunosensor Configuration by EIS. EIS experiments were performed to confirm the immunosensor configuration. The bare SPCE electrode showed a Ret of about 12 kΩ (Figure 3, curve a). The Au modified SPCE showed a much smaller resistance (Ret) compared to the bare electrode (Figure 3, curve b) because the good conductivity of Au NPs could accelerate [Fe(CN)6]3-/4- to the electrode. After further modification with Nb11, the Ret increased (Figure 3, curve c) due to steric hindrance of Nb11. The Ret further increased (Figure 3, curve d) when BSA was added. In the presence of target Apo-A1, the immunoreaction was triggered resulting in subsequent formation of immunocomplex on the sensing interface and hence increased Ret (Figure 3, curve e). Finally, another immunoreaction was 10


Page 10 of 25

Page 11 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


triggered between the Ag-nHAP/Nb conjugate and the target Apo-A1 on the sensing interface, leading to a much larger Ret (Figure 3, curve f) because of the huge steric hindrance. The step-by-step fabrication of the immunosensor is therefore confirmed. Assay Performance. Under the optimum conditions (10 µL of HCl solution, deposition time of 180 s and 20 µL of Au NPs solution), the peak current values of different concentrations of Apo-A1 were recorded. These results were shown in Figure 4A. A linear calibration curve was obtained when plotting current vs the logarithmic value of Apo-A1 concentration from 10-4 to 50 ng mL-1. The regression equation is I=5.70 log c+33.59. Thus the detection limit can be calculated with the equation c = 10(I-33.59)/5.70 (where I is the mean signal of blank measurements +3s, and s is the standard deviation of five replicates of blank measurements). The calculated detection limit was 0.02 pg mL-1. In order to test the assay performance, 10 ng mL-1 of Apo-A1 were selected as an example. Selectivity of the immunoassay was investigated using the modified SPCE electrodes incubating with human immunoglobulin G (HIgG) and BSA as interfering proteins. Both proteins showed a response similar to that of the blank. The mixture of Apo-A1 and HIgG or BSA showed a response similar to that of Apo-A1 at the same concentration (Figure 5A), indicating negligible interference of HIgG and BSA with Apo-A1. Five equally prepared SPCEs were used to investigate reproducibility of the immunosensor by analyzing Apo-A1 solutions of the same concentration. As a result, relative standard deviation (RSD) of 3.5% was obtained (Figure 5B). Therefore, the reproducibility of the immunoassay was good. 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Serum Samples Analysis. As known to all, ELISA is the gold standard of traditional immunoassay. The immunosensor was compared with ELISA by estimating Apo-A1 in human serum samples. As shown in Table S4, compared with ELISA, the RSD of the proposed method was less than 3.9%, which was superior to the best value of ELISA. Using the average measured value obtained by ELISA as the reference value, the relative errors for Apo-A1 detection were less than 6.1%, indicating acceptable accuracy of the assay in analyzing clinical sample. In addition, recovery test for Apo-A1 was also used by adding 1.00, 2.00 and 5.00 mg mL-1 Apo-A1 into the three samples, respectively. These results are listed in Table S5. Recovery rate is between 94.5% and 102%, indicating acceptable precision of the assay.

■ CONCLUSIONS

A novel immunoassay was proposed for ultrasensitive determination of Apo-A1 by loading silver nanoparticles on HAP as label. HAP is an excellent carrier for antibody. Silver nanoparticles loaded HAP (Ag-nHAP) can release silver ions after complete in situ acid dissolution, leaving no interference to the measurement. This strategy simplified the operation procedure and improved the stability. Satisfactory results were obtained when the assay was used to detect Apo-A1. Overall, we see great clinical potential of this immunoassay due to its good sensitivity, wide detection range, simplicity, and good selectivity and reproducibility.

12


Page 12 of 25

Page 13 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


■

ASSOCIATED CONTENT

Supporting Information Available The Supporting Information is available free of charge on the ACS Publications website.

■ AUTHOR INFORMATION

* Corresponding authors: E-mail addresses: [email protected] (Q. Wei), [email protected] (Y. Wan). Author Contributions 1

H.W. and G.L. contributed equally.

Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENT

The study was funded by the National Natural Science Foundation of China (Nos. 21375047, 21175057, 21575050, 21377046, 31271365 and 31471216) and the Special Foundation for Taishan Scholar Professorship of Shandong Province and University of Jinan (No. ts20130937). ■ REFERENCES

(1) Navab, M.; Reddy, S. T.; Van Lenten, B. J.; Fogelman, A. M. Nat. Rev. Cardiol. 2011, 8, 222-232. 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(2) Castelli, W. P.; Garrison, R. J.; Wilson, P. W.; Abbott, R. D.; Kalousdian, S.; Kannel, W. B. Jama 1986, 256, 2835-2838. (3) Santos-Gallego, C. G.; Badimon, J. J.; Rosenson, R. S. Endocrin. Metab. Clin. 2014, 43, 913-947. (4) Toth, P. P.; Barter, P. J.; Rosenson, R. S.; Boden, W. E.; Chapman, M. J.; Cuchel, M.; D'Agostino, R. B., Sr.; Davidson, M. H.; Davidson, W. S.; Heinecke, J. W.; Karas, R. H.; Kontush, A.; Krauss, R. M.; Miller, M.; Rader, D. J. J. Clin. Lipidol. 2013, 7, 484-525. (5) Gafvels, M.; Bengtson, P. Clin. Chim. Acta 2015, 442, 87-95. (6) Lai, G.; Yan, F.; Wu, J.; Leng, C.; Ju, H. Anal. Chem. 2011, 83, 2726-2732. (7) Zhu, C.; Yang, G.; Li, H.; Du, D.; Lin, Y. Anal. Chem. 2015, 87, 230-249. (8) Li, H.; Mu, Y.; Yan, J.; Cui, D.; Ou, W.; Wan, Y.; Liu, S. Anal. Chem. 2015, 87, 2007-2015. (9) Li, H.; Sun, Y.; Elseviers, J.; Muyldermans, S.; Liu, S.; Wan, Y. Analyst 2014, 139, 3718-3721. (10) Saha, K.; Agasti, S. S.; Kim, C.; Li, X.; Rotello, V. M. Chem. Rev. 2012, 112, 2739-2779. (11) Urbanova, V.; Magro, M.; Gedanken, A.; Baratella, D.; Vianello, F.; Zboril, R. Chem. Mater. 2014, 26, 6653-6673. (12) Cai, Y.; Li, H.; Du, B.; Yang, M.; Li, Y.; Wu, D.; Zhao, Y.; Dai, Y.; Wei, Q. Biomaterials 2011, 32, 2117-2123. (13) Xu, W.; Yi, H.; Yuan, Y.; Jing, P.; Chai, Y.; Yuan, R.; Wilson, G. S. Biosens. 14


Page 14 of 25

Page 15 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Bioelectron. 2015, 64, 423-428. (14) Lin, J.; He, C.; Zhang, S. Anal. Chim. Acta 2009, 643, 90-94. (15) Wei, Q.; Li, R.; Du, B.; Wu, D.; Han, Y.; Cai, Y.; Zhao, Y.; Xin, X.; Li, H.; Yang, M. Sens. Actuators, B 2011, 153, 256-260. (16) Jeong, B.; Akter, R.; Han, O. H.; Rhee, C. K.; Rahman, M. A. Anal. Chem. 2013, 85, 1784-1791. (17) Zhang, J.; Lei, J.; Xu, C.; Ding, L.; Ju, H. Anal. Chem. 2010, 82, 1117-1122. (18) Zhang, B.; Liu, B.; Chen, G.; Tang, D. Biosens. Bioelectron. 2015, 64, 6-12. (19) Wei, Q.; Li, T.; Wang, G.; Li, H.; Qian, Z.; Yang, M. Biomaterials 2010, 31, 7332-7339. (20) Fang, Y. S.; Huang, X. J.; Wang, L. S.; Wang, J. F. Biosens. Bioelectron. 2015, 64, 324-332. (21) Hu, C.; Dou, W.; Zhao, G. Electrochim. Acta 2014, 117, 239-245. (22) Wang, H.; Li, X.; Mao, K.; Li, Y.; Du, B.; Zhang, Y.; Wei, Q. Anal. Biochem. 2014, 465, 121-126. (23) Zang, S.; Liu, Y.; Lin, M.; Kang, J.; Sun, Y.; Lei, H. Electrochim. Acta 2013, 90, 246-253. (24) Wang, G.; Gang, X.; Zhou, X.; Zhang, G.; Huang, H.; Zhang, X.; Wang, L. Talanta 2013, 103, 75-80. (25) Yang, F.; Yang, Z.; Zhuo, Y.; Chai, Y.; Yuan, R. Biosens. Bioelectron. 2015, 66, 356-362. (26) Zhang, B.; Liu, B.; Liao, J.; Chen, G.; Tang, D. Anal. Chem. 2013, 85, 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

9245-9252. (27) Qian, J.; Zhang, C.; Cao, X.; Liu, S. Anal. Chem. 2010, 82, 6422-6429. (28) Chen, L.; Zeng, X.; Si, P.; Chen, Y.; Chi, Y.; Kim, D. H.; Chen, G. Anal. Chem. 2014, 86, 4188-4195. (29) Rashidi Nassab, H.; Souri, A.; Javadian, A.; Amini, M. K. Sens. Actuators, B 2015, 215, 360-367. (30) Lee, S.; Bong, S.; Ha, J.; Kwak, M.; Park, S.-K.; Piao, Y. Sens. Actuators, B 2015, 215, 62-69. (31) Grabarczyk, M.; Wasag, J. Talanta 2015, 139, 132-137. (32) Zhang, S.; Du, B.; Li, H.; Xin, X.; Ma, H.; Wu, D.; Yan, L.; Wei, Q. Biosens. Bioelectron. 2014, 52, 225-231. (33) Lai, G.; Wu, J.; Ju, H.; Yan, F. Adv. Funct. Mater. 2011, 21, 2938-2943. (34) Kokkinos, C.; Economou, A.; Petrou, P. S.; Kakabakos, S. E. Anal. Chem. 2013, 85, 10686-10691. (35) Kokkinos, C.; Economou, A.; Speliotis, T.; Petrou, P.; Kakabakos, S. Anal. Chem. 2015, 87, 853-857. (36) Nakane, P. K.; Kawaoi, A. J. Histochem. Cytochem. 1974, 22, 1084-1091.

16


Page 16 of 25

Page 17 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure captions Scheme 1. Workflow of selecting anti-Apo-A1 Nbs from an immunized Bactrian camel.

Scheme 2. Fabrication and working principle of the nanobody-based electrochemical immunoassay for sensing of Apo-A1.

Figure 1. (A) Equilibrium dissociation constant values between Apo-A1 and two Nbs. Apo-A1 solutions were injected at concentrations of 27, 81 and 243 nmol L-1 (C3 to C1). (B)Thermal stability of Nb11 and Nb19. Anti-Apo-A1 Nbs were incubated at different temperatures (37 ºC, 70 ºC, 90 ºC) for various periods of time (0.5, 1, 2 h). Three independent biological replicates were performed for test.

Figure 2. (A) The LSSV responses of the conjugate after (a) and before (b) acid dissolution with 10 µL of 10% (V/V) HCl solution. Optimization of the acid volume (B), the deposition time (C) and the volume of Au NPs solution (D).

Figure 3. EIS measurements for each step of the fabrication procedure of the immunosensor. Figure 4. Calibration curve of the immunosensor at the concentrations of 10-5, 10-4, 10-3, 0.01, 0.05, 0.1, 1.0, 10 and 50 ng mL-1.

Figure 5. (A) Electrochemical responses for blank control, Apo-A1, HIgG, BSA, the mixture of Apo-A1 and HIgG or BSA. Error bars are the mean standard deviation of three replicates. (B) Parallel experiments using five equally prepared SPCEs for the analysis of 10 ng mL-1 Apo-A1.

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1

18


Page 18 of 25

Page 19 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 2

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1

20


Page 20 of 25

Page 21 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3

22


Page 22 of 25

Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5

24


Page 24 of 25

Page 25 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC only

25


Nanobody-Based Electrochemical Immunoassay ... - ACS Publications

Recommend Documents