Nanobody-Based Apolipoprotein E Immunosensor for Point-of-Care

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Nanobody-based Apolipoprotein E Immunosensor for Point-of-Care Testing Xiang Ren, Junrong Yan, Dan Wu, Qin Wei, and Yakun Wan ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00495 • Publication Date (Web): 08 Sep 2017 Downloaded from http://pubs.acs.org on September 9, 2017

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Nanobody-Based Apolipoprotein E Immunosensor for Point-of-Care Testing Xiang Ren†#, Junrong Yan§#, Dan Wu†, Qin Wei†*, Yakun Wan‡* †

Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, Shandong, 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, and §Institute of Life Sciences, Southeast University, Nanjing 210000, P.R. China

ABSTRACT: Alzheimer′s disease (AD) biomarkers can reflect the neurochemical indicators which is used to estimate the risk in clinical nephrology. Apolipoprotein E (ApoE) is an early biomarker for AD in clinical diagnosis. In this research, through bactrian camel immunization, lymphocyte isolation, RNA extraction and library construction, ApoE-specific Nbs with high affinity were successfully separated from an immune phage display nanobody library. Herein, a colorimetric immunosensor was developed for the point-of-care testing of ApoE by layer-by-layer nano-assembly techniques and novel nanobodies (Nbs). Using highly oriented Nbs as the capture and detection antibodies, an on-site immunosensor was developed by detecting the mean gray value of fade color due to the glutaraldehyde@3-aminopropyltrimethoxysilane oxidation by H2O2. The detection limit of AopE is 0.42 pg/mL, and the clinical analysis achieves a good performance. The novel easy-operated immunosensor may have potential application in the clinical diagnosis and real-time monitoring for AD. KEYWORDS: nanobody, apolipoprotein E, point-of-care testing, colorimetric, immunosensor

A

lzheimer′s disease (AD) biomarkers are neurochemical indicators used to estimate the risk or presence of this disease, which affects currently more than three million people in Europe and constitutes thus a major societal problem.1 Apolipoprotein E (ApoE) is a major genetic risk factor for AD and it can lead to an excess amyloid formation in the brain.2 Thus, to develop an effective method to detect the ApoE is essential and important. In the middle 1990s, researchers found camels possessing a unique class of unconventional antibodies naturally devoid of the light chains in their sera,3 called heavy-chain only antibodies (HCAbs). The N-terminal variable domains of HCAbs stand for the smallest natural occurring antigen binding fragments known,4 termed as the variable domain of heavy chain of the heavy chain-only antibody (VHH) or Nanobody® (Nb). Nanobodies (Nbs) have a molecular size of 12-15 kDa that is distinctly smaller than the classical immunoglobulins G molecule (IgG) (~160 kDa), antigenbinding fragment (Fab) (~50 kDa) and single-chain variable fragment (scFv) (~27 kDa). These monomeric antibody fragments exhibit several unique advantages in terms of high affinity,5 ease of cloning and expression in Escherichia coli (E. coli) and Saccharomyces cerevisiae,6 high thermal, chemical and conformational stabilities,7 more solubility, and ease of generating bispecific or multimeric constructs.8 Besides, these entities show an extended complementarity determining region (CDR) 3 that enables them to recognize haptens and cryptic epitopes that are less accessible for conventional antibodies.9 The good thermal, chemical, and conformational stabilities bring about greater resistance to the denaturation, degradation, or regeneration conditions in immunoassays. The single-domain nature of the VHHs facilitates tagging with

single chemical reactive groups for directional coupling on a biosensors surface.10 All these distinctive advantageous properties make VHHs emerge as highly attractive novel candidates in immunoassays applications.5,11,12 Recently, portable sensing devices have been developed remarkably like paper-based analytical devices,13 agar powder test strip,14 and microwell plate array.15 The researchers gained great interest on these devices due to their obvious advantages, such as low cost, easy operation and real-time monitoring.13 Compared with the reported point-of-care testing (POCT) or other immunosensors,16–24 the indium tin oxide (ITO) based POCT can reveal the similar results without the complicated analytical device preparation. The colorimetric sensing strategy can quantify the analyte through color change. Traditional immunosensors cannot detect an analyte through naked eyes, and the quantification is through spectrophotometry which is not suitable for on-site analysis. Herein, a camera was employed. The picture can be translated into a gray value by software which is related to the detection target molecule concentration. Thus, it is easy to operate and has the potential application in the point-of-care analysis in undeveloped countries (the cost is shown in SI). On the basis of above considerations, nanobody and ITO based POCT immunosensor can achieve good performance, however, has not been reported. Here, we report a colorimetric immunosensor for the detection of ApoE. The anti-ApoE Nbs were acquired from camels, and the high specificity and affinity of the Nb make it a better candidate in immunosensor. The sensing system was fabricated by the 3-aminopropyltrimethoxysilane (APTMS) and glutaraldehyde (GA). The resulting complex (APTMSGA) exhibited brick-red color and could be oxidized by H2O2,

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leading to a fading color of the brick-red. The relative gray value can be quantified by the software of Image J. The detection limit of AopE is 0.42 pg/mL, and the clinical analysis achieves a good performance. The novel easy-operated immunosensor may have potential in the clinical diagnosis and real-time monitoring for AD.

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high purity (Figure 2B). Meanwhile, milligram quantities of Nbs were yielded (Table S1).

RESULTS AND DISCUSSION Library construction. To generate ApoE-specific Nbs, a young and healthy camel was immunized with ApoE (Details in SI). The VHH genes were amplified from cDNA that reverse transcribed from lymphocyte RNA, and the first PCR fragments have apparent bands at about 700 bp (Figure 1A). The VHH-only fragments were amplified with the 700 bp fragments as the templates and its final bands size is around 400 bp (Figure 1A). After the library was successfully constructed, we determined the library capacity and the correct insertion ratio. According to the results of gradient dilutions (Figure S2), the library size reached to 2.25×109 colonyforming units (CFU). Meanwhile, as shown in Figure 1B and 2C, the correct insertion ratio was nearly 83.3%. The library capacity was significantly large enough for us to isolate the specific Nbs against ApoE protein. Overall, we have successfully generated a phage-displayed immune VHH library for the following selection of anti-ApoE Nbs.

Figure 2. Isolation of anti-ApoE Nbs. (A) The anti-ApoE Nbs were classified into five families according to the significantly different amino acid sequences in CDR3 regions, named Nb05, 19, 21, 24 and 40. Amino acids positions of the framework region (FR) and of the three CDRs are numbered according to the IMGT Scientific chart for the V-Domain and are indicated at bottom. (B) The five ApoE-specific Nbs were expressed as C-terminal HA-tag and His6tag proteins. The purity of these Nbs was analyzed by SDS-PAGE.

SPR analysis. As we needed to use Nb05 and Nb40 for the further ApoE detection, we were motivated to determine the affinity between the Nbs and ApoE. We measured the affinities by SPR analysis using PlexArray® HT system. As the sensorgrams (Figure 3A) showed, these two Nbs presented significantly high affinity to ApoE with their equilibrium dissociation constants (KD) of 3.40×10-9 and 8.14×10-10 M, respectively (Figure 3B). The high affinity of the paired Nbs (Figure 3C) provided a serious possibility for further biosensor development and diagnostic application.

Figure 1. Construction of the ApoE-specific VHH library. (A) The schema graph of VHH genes cloning by the two-step nested PCR. (B) The PCR products were run on agarose gel. The band around 700 bp produced by the first PCR (Left) and the band around 400 bp produced by the secondary PCR (Right) were re-extracted by gel purification. (C) 24 colonies were randomly selected to determine the correct insertion ratio of VHH genes by PCR.

Anti-ApoE Nbs identification. Herein, we identified antiApoE Nbs using phage display technology. We calculated the relative enrichment of phage particles eluted from the wells coated with ApoE versus those without ApoE. The enriching times dramatically increased from 18 to 737.5-fold in the third round of bio-panning (Figure S3), which meant we had a good chance of obtaining ApoE-specific Nbs with high specificity. Next, 95 individual colonies were randomly picked to identify ApoE-specific Nbs. After DNA sequencing, there were twelve Nbs containing a different amino acids sequence in CDR3 regions. Furthermore, among these twelve Nbs, five were chosen as the representative families of ApoE-specific Nbs as they exhibited significantly diverse amino acid sequences in CDR3 regions (Figure 2A). Nbs expression and purification. The recombinant phagemids encoding anti-ApoE Nbs were transformed from TG1 to WK6 cells and the Nbs were expressed as soluble Cterminally HA and His6-tagged proteins. The His6 oligopeptide was designed as the tag for purification and the HA tag could be used as the recognition component in ELISA identification of ApoE-specific Nbs. The fusion proteins were purified on a His-Select column and analyzed by SDS-PAGE. SDS-PAGE analysis demonstrated we obtained good quality of Nbs with

Figure 3. Affinity analysis. (A, B) The affinities between ApoE and the five Nbs were determined by SPRi binding assay. ApoE dilutions were injected at concentrations of 1, 3, 9, 27 and 81 nM. (C) Relevant parameters of the kinetic analysis.

Preparation of ApoE immunosensor. The fabricated immunosensor is in sandwich type. The labels were fabricated by Au-TiO2 (Details in SI), GOD, and anti-ApoE Nbs (Details in SI). Specifically, 1 mg Au-TiO2 was added to 1 mL of ultrapure water under magnetic stirring to form a uniform dispersion. Then, 500 µg GOD and 500 µg anti-ApoE Nb were added in the Au-TiO2 dispersion. The mixture was mildly shaking at 4 °C for 24 h to form a firm compound. The compound was centrifuged to remove the unbound biomolecules. The remaining compound was redispersed in 1 mL of ultrapure water to form the labels (AuTiO2@GOD@anti-ApoE Nb05) (Figure 4). In this labels, AuTiO2 was the amplifier to enlarge the loading capacity. GOD was used as the catalysis to act on the glucose and anti-ApoE Nb05 was used as the detection antibody for the immunosensor fabrication.

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The sensor base (ITO/APTMS-GA/IL-CS/Au NPs) was ready for the immunosensor. First, anti-ApoE Nb40 (6 µL) was dropped on the surface of Au NPs to form a stable layer because of the strong interaction between Au and -NH2. Then 3 µL of BSA was incubated on the sensor to eliminate the nonspecific active sites. The immunosensor was washed slightly with PBS to remove the unbound biomolecules. Next, various concentrations of ApoE were incubated on the former layer. Last, the labels (Au-TiO2@GOD@anti-ApoE Nb05) were incubated on the immunosensor. The immunosensor was well fabricated (ITO/APTMS-GA/IL-CS/Au NPs/anti-ApoE Nb40/BSA/ApoE/Au-TiO2@GOD@anti-ApoE Nb05) (Figure 4). The catalytic reaction was proceeded when the glucose was dropped on the immunosensor surface. The ApoE concentrations were acquired due to the different color fade intensity.

µg/mL range for serum,25 the serum was diluted (1:10000) with PBS before testing. The matrix effects can be neglected without introducing obvious differences in the highly diluted samples.26 The recoveries for the ApoE in human serum samples ranged from 94.0% to 103%, which demonstrated the acceptable accuracy.

Figure 5. Calibration curve of the ApoE. (Error bar = SD, n = 5)

Figure 4. Fabrication of the immunosensor for ApoE.

Detection of ApoE. For AD detection, an ApoE POCT immunosensor (Figure 4) was fabricated using anti-ApoE Nbs. The labels of Au-TiO2@GOD @anti-ApoE Nb05 can research a well result due to the combination of Ti-O bond and flavin adenine dinucleotide (FAD) from the GOD (Figure S6), indicating the loading of GOD was through the Au-NH2 and Ti-O interaction. 6 µL of (10 mM) glucose was added on the modified ITO for adequate reaction until the color intensity changed. Glucose can be catalyzed by GOD to generate H2O2 which can oxidize the dimer to fade the color intensity. Each ITO was calculated by the software of Image J to measure the quantity of ApoE. The linear relationship between the mean gray value and ApoE concentration was from 1.0 pg/mL to 10 ng/mL (Figure 5). The calibration curve is Y = 90.76 + 6.98 X (R2 = 0.9973) with a detection limit of 0.42 pg/mL. The specificity of the proposed ApoE immunosensor was investigated by measuring the mean gray value of BSA, AFP, CEA and PSA at a concentration of 10 ng/mL, which is 10fold to ApoE (Figure S7). The four interference biomoleculars cannot affect the obvious difference of gray values but the pure ApoE can arrive at an excellent result which is consistent with previous calibration curve. The reproducibility of the colorimetric immunosensor was also discussed in the research. Five prepared immunosensors were tested under the identical condition, and the relative standard deviation (RSD) of the measurement was 4.7%, suggesting that the precision of the immunosensor was reasonably good for the detection of ApoE. The storage stability was also investigated. Several prepared immunosensors were stored in the refrigerator at 4 ºC for one month, the mean grey values were the same as the previous values indicating that the results were satisfactory and acceptable. Clinical diagnose. Real sample analysis was also studied to evaluate the colorimetric immunosensor property which was shown in Table S2. Due to the levels of ApoE typically in the

Meanwhile, ELISA kit was also conducted to evaluate the proposed colorimetric immunosensor. The ApoE concentration of the diluted human serum sample was tested five times by ELISA kit, respectively. The results are shown in the Table S3. Based on the F-test, the calculated F value is less than the theoretical one, demonstrating the precisions of these two methods are highly equivalent. By t-test analysis, the mean values were not obviously different from the data gained from ELISA kit, showing the system error can be ignored. Through the F-test and t-test, the precision and accuracy can be ensured.

CONCLUSIONS In this work, an ITO-based point-of-care colorimetric immunosensor was developed for the detection of ApoE based on camel single-domain Nbs. The simplicity, good thermostability, high specificity, and affinity of Nb make it a highly attractive candidate in biosensor application. The naked eye observation can realize the accuracy detection with a camera or even a cell phone, so it would provide a promising technique for other biomarkers detection in undeveloped countries.

ASSOCIATED CONTENT Supporting Information Figure S1 – Figure S7; Table S1 – S3; Experimental sections. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]

ORCID Qin Wei: 0000-0002-3034-8046

Notes # X. Ren and J. Yan contributed equally.

ACKNOWLEDGMENT This work was supported by the National Key Scientific Instrument and Equipment Development Project of China (No. 21627809), the National Natural Science Foundation of China (Nos. 21375047, 21575137, 21575050, 21601064), the Special Foundation for Taishan Scholar Professorship of Shandong Province (No. ts20130937), the Science and Technology Devel-3

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opment Plan of Shandong Province (No. 2014GSF120004), and Graduate Innovation Foundation of UJN (YCXB15004). (15)

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