Dual-Channel Surface Plasmon Resonance for Quantification of ApoE

Oct 17, 2018 - ... Lambert-Hammill, M.; Wierzbicki, A. S. Simple sequence-specific-primer-PCR method to identify the three main apolipoprotein E haplo...
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Dual-Channel Surface Plasmon Resonance for Quantification of ApoE Gene and Genotype Discrimination in Unamplified Genomic DNA Extracts Xinyao Yi,† Yonghong Xia,† Binrong Ding,‡ Ling Wu,† Shengqiang Hu,† Zixiao Wang,† Minghui Yang,† and Jianxiu Wang*,† †

College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, People’s Republic of China 410083 Department of Geriatrics, The Third Xiangya Hospital of Central South University, Changsha, Hunan, People’s Republic of China 510060

ACS Sens. Downloaded from pubs.acs.org by UNIV OF SOUTH AUSTRALIA on 10/28/18. For personal use only.

‡

ABSTRACT: Identification of gene variation is of great importance for attaining information related to disease susceptibility. A highly sensitive and specific surface plasmon resonance (SPR) method for quantification of the apoE gene and genotype discrimination was demonstrated. The complementary sequences with the specific recognition sites of GCGC bases upon hybridization to the preimmobilized biotinylated probes could be cleaved by the restriction enzyme HhaI, while the existence of the single-base mismatch (GTGC) prevented the cleavage reaction. In both cases, the incorporation of streptavidin increased the sensitivity of the SPR assay, and the detection levels of 10 fM and 50 fM for the complementary and single-base mismatched sequences were attained, respectively. The sensing protocol is simple, label-free, and quantitative, thus avoiding the complicated polymerase chain reaction (PCR) amplification procedures. The proposed method serves as a viable means for facile and sensitive analyses of apoE genes in four unamplified genomic DNA extracts. KEYWORDS: surface plasmon resonance, apo E gene, genotyping, restriction enzyme HhaI, Alzheimer’s disease than that with ε3/3.3 The apoE genotyping could provide useful information on AD precaution and diagnosis. Typically, traditional apoE genotyping protocols rely on hybridization-based genetic amplification, such as allele-specific polymerase chain reaction (PCR),4,6 primer extension,7 and gene amplification and cleavage.8 These methods usually involve tedious assay procedures, longer assay time, and highly trained operators, which limits their practical genotyping applications. Microarray offers multiplexed and parallel identification of apoE genotyping7,9 but usually suffers from lower sensitivity and cross-reactivity.10,11 Electrochemical methods are simple, rapid, and sensitive12 and have been widely used in apoE genotyping.13−15 Based on the modified Randles’ equivalent circuit, discrimination of the singlenucleotide mismatch in apoE alleles has been performed by comparing the difference in the charge transfer resistance before and after Ni2+ addition.13 DNA electrochemical biosensors have been developed for the detection of apoE genotypes in PCR-amplified DNA extracted from human blood.14 By measuring microfluidic electrochemical melting

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ene polymorphisms may alter the structure and function of the coded proteins, thus increasing the risk of disease.1 Discrimination of the genotypes is essential for illustrating the influence of genetic variations, providing scientific information on disease precaution, clinical diagnosis, and personalized medication.1 Apolipoprotein E (apoE) is an important component of plasma lipoproteins, serving as a major cholesterol carrier that supports lipid transport.2 The polymorphic alleles of apoE have been demonstrated to be the main genetic determinants of Alzheimer’s disease (AD).3 The apoE isoforms (alleles ε2, ε3, and ε4) possess two polymorphic sites in the codons of 112 and 158 on the gene located in chromosome 19 (allele ε2 has a thymine base at codons 112 and 158; ε3 has a thymine base at codon 112 and a cytosine base at codon 158; ε4 has a cytosine base at the both codons) and are combined into six genotypes including three homozygous ones (ε2/2, ε3/3, and ε4/4) and three heterozygous ones (ε2/3, ε2/4 and ε3/4).4 Among them, the genetic risk factor of the ε4 allele is strongly related to AD, and the risk of AD increases in individuals with one or two copies of the ε4 allele as compared to that with ε3/3.3,5 Conversely, the ε2 allele exerts a protective effect on AD, and the risk of AD in individuals with apoE ε2/2 or ε2/3 is lower © XXXX American Chemical Society

Received: August 15, 2018 Accepted: October 17, 2018 Published: October 17, 2018 A

DOI: 10.1021/acssensors.8b00845 ACS Sens. XXXX, XXX, XXX−XXX

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ACS Sensors curves, homozygous and heterozygous apoE genotypes were differentiated.15 A piezoelectric biosensor for the detection of apoE gene polymorphism in clinical samples of PCR-amplified DNA was also constructed.16 However, the above methods for apoE genotyping were either conducted at a higher temperature15 or involved PCR-amplified DNA samples.14,16 To realize direct genotyping and apoE quantification from genomic DNA samples without pre-enrichment, the development of highly sensitive and specific methods is still challenging. As an optical technique, surface plasmon resonance (SPR) is highly sensitive to the refractive index or thickness change at the metal surface, which enables label-free and real-time monitoring of the biomolecular interactions.17−19 Due to the capability for multiplexed analysis, surface plasmon resonance imaging (SPRi) has been widely used for the assay of DNA and single nucleotide polymorphisms.20−23 With the aid of Au nanoparticle tags, more than 1000-fold improvement in the sensitivity for the assay of target oligonucleotide was achieved by SPRi, and the detection limit was estimated to be 10 pM.20 Via combination of surface enzymatic ligation and nanoparticle amplification, the detection limit for the assay of single nucleotide polymorphism of the BRCA1 gene associated with breast cancer was estimated to be 1 pM by SPRi.21 An SPRi setup was applied to the detection of gene mutations in genetic disease cystic fibrosis with a detection limit of 7 fmol/mm2.22 Using neutralized chimeric DNA probes, sensitive SPRi detection of single nucleotide polymorphisms has been achieved.23 Despite the multiplexing capability of SPRi, the CCD camera compromised the sensitivity of the method. As a result, it still remains a challenge to develop highly sensitive SPR-based methods for DNA and single nucleotide polymorphism detection. In this study, quantification of apoE gene and genotyping discrimination based on the enzymatic cleavage reaction has been conducted using a dual-channel SPR. The allele-specific biotinylated DNA probes were anchored on the sensor chips for recognition of the fragments of the apoE gene with complementary and single-base mismatched sequences. The duplex with GCGC base pairs could be cleaved by the restriction enzyme HhaI, while the presence of the single-base mismatch (GTGC) hindered the cleavage reaction. In the two cases, the biotinylated fragments residing on the surface were recognized by streptavidin (SA), leading to different SPR signals. The apoE genotyping in four unamplified genomic DNA extracts was achieved.

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Table 1. Sequences of the Fragments of apoE Isoforms and the Abbreviations

to the fragments of allele ε4 at codon 112 (Tc 1) and those of allele ε3 or ε4 at codon 158 (Tc 2), respectively, which ensures the specific cleavage of the GCGC base pairs in the DNA duplex by restriction enzyme HhaI. The enzyme recognition regions are in bold, and the mismatched bases are underlined. The restriction enzyme HhaI was obtained from Takara Bio. Inc. (Dalian, China). All the reagents were of analytical grade and used without further purification. Unless otherwise stated, all the stock solutions were prepared daily with deionized water treated with a water purification system (Simplicity185, Millipore Corp, Billerica, MA). Solution Preparation. The DNA probes were prepared with phosphate buffered saline (PBS, 10 mM phosphate, 10 mM NaCl, pH 7.4), and the targets were dissolved in PBS containing 5 mM MgCl2. MUA and EA were dissolved in ethyl alcohol and water, respectively. EDC/NHS solution was prepared by mixing 0.4 M EDC and 0.1 M NHS in water before the activation of MUA self-assembled monolayers (SAMs). The restriction enzyme HhaI was diluted with the buffer (pH 7.5) containing 10 mM Tris-HCl, 100 mM MgCl2, 10 mM dithothreitol, and 500 mM NaCl. SA was diluted to 20 nM with PBS before use. SPR Detection. The SPR assay was performed on a BI-SPR 4000 system (Biosensing Instrument Inc., Tempe, AZ). Au films coated onto BK7 glass slides were annealed in a hydrogen flame to eliminate surface contaminants. The annealed Au films were immersed in 0.5 mM MUA solution for 24 h and the SAMs were gently rinsed with ethanol, water, and dried under nitrogen. The carboxylic acid groups on the MUA SAMs were then activated with a mixed solution containing 0.4 M EDC and 0.1 M NHS for 30 min, followed by dropping a 100 μL aliquot of 1 μM aminated DNA probes with a biotin tag. The films were kept in a humidity chamber for 1 h, and after rinsing with water and drying under nitrogen, they were treated with 1 M EA to block the empty sites. DNA targets with various sequences were hybridized to the anchored probes in PBS containing 5 mM MgCl2 at room temperature for 3 h. After that, 100 U/mL of HhaI was cast onto the sensor chips at 37 °C for 3 h to clip the duplex DNA at the cleavage site. The melting points of probe 1 and probe 2 were deduced to be 76.2 and 71.7 °C, respectively. To amplify the SPR signals, SA was flowed over the sensor chips at a flow rate of 20 μL/min. DNA Extraction. The unamplified genomic DNA extracts were obtained from the blood of four donors using the TIANamp Genomic DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. The volume of the obtained DNA extracts was 50 μL, and that of the blood samples used was 2 mL.

EXPERIMENTAL SECTION

Chemicals and Reagents. 11-Mercaptoundecanoic acid (MUA), ethanolamine (EA), K2HPO4, KH2PO4, NaOH, MgCl2, N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and streptavidin (SA) were acquired from Sigma (St. Louis, MO). The oligonucleotides were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). The sequences of apoE ε2, apoE ε3, and apoE ε4 that surround codons 112 and 158 are shown in Table 1. Tc 1 (complementary target 1) was designed as the fragment of allele ε4 at codon 112. Tc 2 (complementary target 2) denotes the fragment of allele ε3 or ε4 at codon 158. Tsm 1 (single mismatched target 1) is the fragment of allele ε2 or ε3 at codon 112, and Tsm 2 (single mismatched target 2) denotes the fragment of allele ε2 at codon 158. Biotinylated probes 1 and 2 possess sequences of 5′-NH2-(CH2)6-TTTTTCAGGCGGCCGCGCACGTC-biotin-3′ and 5′-NH2-(CH2)6TTTTTCACTGCCAGGCGCTTCTG-biotin-3′, respectively. The sequences of probes 1 and 2 were designed to be complementary

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RESULTS AND DISCUSSION The schematic of quantification of apoE gene and genotype discrimination is illustrated in Figure 1. The biotinylated DNA probes 1 and 2 were tethered onto the MUA SAMs (CH1 and CH2, respectively) through EDC/NHS cross-linking chemistry. After hybridization to their respective complementary B

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Figure 1. Schematic of quantification of apoE gene and genotype discrimination by SPR. The fluidic channels 1 and 2 (CH1 and CH2) were preimmobilized with the biotinylated probes 1 and 2, respectively. After hybridization to their respective targets (CH1 and CH2 in the top panel for the complementary strands of Tc 1 and Tc 2, respectively, and those in the bottom panel for the strands of Tsm 1 and Tsm 2 with a single-base mismatch, respectively), HhaI was dropped onto the sensor chips, and the cleavage reaction occurred in the case of the complementary strands of Tc 1 and Tc 2. The injection of 20 nM SA capable of recognizing the biotinylated fragment was used to amplify the SPR signals.

targets (Tc 1 and Tc 2 to the biotinylated probe 1 and 2, respectively), the GCGC base pairs in the DNA duplex could be selectively cleaved by HhaI. The detachment of the biotinylated fragment from the surface hinders the attachment of SA with a large molecular weight of 60 kDa,24 leading to smaller SPR signals (top panel). However, the existence of a single-base mismatch in the enzyme recognition sites (GTGC in Tsm 1 and Tsm 2) prevents the enzymatic cleavage reaction, and the biotinylated fragment was retained on the sensor chips. The attachment of SA thus amplifies the SPR signals (bottom panel). The binding affinity between the biotin tag and SA is high, comparable to that of the covalent bond.25 By examining the difference in the SPR signals, allele-specific apoE genotyping could be conveniently achieved. The injection of 20 nM SA onto the sensor chips preimmobilized with the unbiotinylated DNA probes after hybridization to their respective complementary targets (curve a in Figure 2A and B) did not produce a detectable SPR signal, indicating that the nonspecific adsorption of SA on the duplex DNA-covered films is negligible. However, the attachment of SA to the biotinylated duplex DNA produced large SPR signals of 300 mDeg (curve b in Figure 2A) and 200 mDeg (curve b in Figure 2B). The difference in the SPR signals in curve b of Figure 2A and B might be ascribed to the difference in the hybridization efficiency of probe 1/Tc 1 and probe 2/Tc 2, and in the probe amination and biotinylation. In the presence of HhaI, HhaI could selectively cleave the GCGC base pairs in the duplex DNA, detaching the biotinylated fragments from the surface, and the SPR signals of 40 mDeg (curve c in Figure 2A) and 30 mDeg (curve c in Figure 2B) were attained upon injection of 20 nM SA. The detectable SPR signals in curve c of Figure 2A and B were the result of the incomplete cleavage of the duplex DNA. In the case of Tsm 1 or Tsm 2 with a single-base mismatch (curve d in Figure 2A and B), similar SPR signals to those in curve b of Figure 2A and B were obtained, indicating that the single-base mismatch on the

Figure 2. SPR sensorgrams upon injection of 20 nM SA onto the fluidic channels (A) after hybridization of the unbiotinylated probe 1 to its complementary strand of Tc 1 (a), the biotinylated probe 1 to Tc 1 (b, c), or Tsm 1 with a single-base mismatch (d) and (B) after hybridization of the unbiotinylated probe 2 to its complementary strand of Tc 2 (a), the biotinylated probe 2 to Tc 2 (b, c), or Tsm 2 with a single-base mismatch (d). The concentration of the probes and targets was maintained at 1 μM. In panels A and B, curves a and b show the cases in the absence of HhaI, while curves c and d depict the cases in the presence of HhaI.

cleavage sites prevents the cleavage reaction and leaves the biotinylated duplex DNA intact. The remarkably different SPR signals in curves c and d in Figure 2A and B suggest that the method serves as a viable means for distinguishing complementary and single base-mismatched DNA strands and for apoE genotyping. The SPR signals decreased with the increasing concentrations of HhaI between 20 U/mL and 100 U/mL and plateaued beyond 100 U/mL. A similar trend for the cleavage time was also attained, and beyond 3 h, the SPR signals began to level off. Thus, the optimal HhaI concentration and cleavage time were fixed at 100 U/mL and 3 h, respectively (data not shown). Figure 3 depicts the dependence of the SPR signals on the concentrations of the complementary strands of Tc 1 (A) and Tc 2 (B). The increase in the target concentrations leads to the formation of more duplex DNA, which increases the tendency for the cleavage reaction. The insets show the linear portions of C

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Figure 4. SPR sensorgrams upon injection of 20 nM SA onto the fluidic channels after cleavage of the hybrids formed via (A) hybridization of the biotinylated probe 1 to (a) 10 nM complementary strand of Tc 1, (b) 10 nM Tc 1 + 10 nM Tsm 1 with a single-base mismatch, (c) 10 nM Tc 1 + 10 nM Tc 2 and (B) hybridization of the biotinylated probe 2 to (a) 10 nM complementary strand of Tc 2, (b) 10 nM Tc 2 + 10 nM Tsm 2 with a single-base mismatch, (c) 10 nM Tc 2 + 10 nM Tc 1.

Figure 3. Dependence of the SPR signals on the concentrations of the complementary strands of Tc 1 (A) and Tc 2 (B). The enzymatic cleavage reaction was followed by the injection of 20 nM SA. The absolute errors deduced from three replicated measurements are shown as the error bars. The insets show the linear portions of the curves between 10 fM and 1 pM.

the calibration curves with target concentrations ranging from 10 fM to 1 pM. The linear regression equations were expressed as Signal (mDeg) = −74.1 [Tc 1] (pM) + 282 (R2 = 0.99) (Figure 3A) and Signal (mDeg) = −44.8 [Tc 2] (pM) + 184 (R2 = 0.96) (Figure 3B). The proposed method for the target assay is highly reproducible, as the relative standard deviation values for three replicated measurements are all below 8%. The detection level of Tc 1 or Tc 2 is 10 fM, being much lower than those achievable by the SPR methods with the aid of Au nanoparticle tags (10 pM)20 and enzymatically modified Au nanoparticles (500 fM).26 Such a concentration level is also comparable with those by the SPR assay based on Au nanoparticle-enhanced diffraction grating (10 fM)27 and electromechanical signal transduction (10 fM).28 The sensing protocol is simple and sensitive, holding great promise for assaying the apoE gene in clinical samples. As discussed above, the proposed method is capable of distinguishing complementary and single-base mismatched DNA strands, providing the feasibility for apoE genotyping. For the assay of single-base mismatched DNA strands, the competitive hybridization reaction was performed. The factors contributing to the competitive hybridization beyond the concentrations of target single nucleotide polymorphisms have been described. For example, for performing the competitive hybridization reaction, the concentration of the complementary targets was fixed at 10 nM due to the plateau of the SPR signals at 10 nM Tc 1 or Tc 2 in Figure 3. Furthermore, the hybridization time of 3 h is long enough for the competition of the complementary sequence with the single mismatched sequence for the preimmobilized biotinylated probe at room temperature. As shown in Figure 4, the hybridization of the biotinylated probe 1 to 10 nM Tc 1 (curve a in Figure 4A) and that of the biotinylated probe 2 to 10 nM Tc 2 (curve a in Figure 4B) result in SPR signals of 73 and 32 mDeg, respectively. While mixing a 10 nM complementary strand (Tc 1 or Tc 2) with a 10 nM single-base mismatched strand (Tsm 1 or Tsm 2), the single-base mismatched strand was allowed to compete with the complementary strand for the preimmobilized biotinylated probe 1 or 2, which prevents HhaI from cleaving the GCGC base pairs in the duplex. Hence, an increased SPR signal was attained (160 mDeg in curve b of Figure 4A and 120 mDeg in curve b of Figure 4B). In the case of Tc 2 or Tc 1 (11-base difference relative to Tc 1 or Tc 2, respectively) that was introduced to compete with Tc 1 or Tc 2 for hybridization with the biotinylated probe 1 or 2, respectively (curve c in Figure 4A and B), almost the same SPR signals as those in curve a of Figure 4A and B were

obtained. The rationally designed biosensor thus provides the possibility for quantification of different apoE genotypes. Relying on the competitive hybridization reaction, the dependence of the SPR signals on the concentrations of Tsm 1 (A) and Tsm 2 (B) was illustrated in Figure 5. The increase

Figure 5. Dependence of the SPR signals on the concentrations of Tsm 1 (A) and Tsm 2 (B) with a single-base mismatch. Panel A shows the competitive hybridization between Tsm 1 and 10 nM Tc 1 for preimmobilized probe 1, while panel B depicts the competitive hybridization between Tsm 2 and 10 nM Tc 2 for preimmobilized probe 2. The competitive hybridization reaction was followed by the enzymatic cleavage of the GCGC base pairs in the duplex. The absolute errors deduced from three replicated measurements are shown as the error bars. The insets show the linear portions of the curves between 50 fM and 1 pM.

in the concentrations of Tsm 1 or Tsm 2 with a single-base mismatch suppressed the enzymatic cleavage reaction, leading to larger SPR signals. The insets show the linear portions of the calibration curves between 50 fM and 1 pM. The linear regression equations were expressed as Signal (mDeg) = 26.7 [Tsm 1] (pM) + 73.4 (R2 = 0.98) and Signal (mDeg) = 21.6 [Tsm 2] (pM) + 33.2 (R2 = 0.99). The detection level for the single-base mismatched strand (50 fM) was higher than that for the complementary strand (10 fM), which was attributed to the weaker hybridization capability between the mismatched strands and the preimmobilized probes.29 However, such a concentration level (50 fM) for the detection of single-base mismatch is much lower than those by Au nanoparticleenhanced SPR imaging (1 pM)21 and triple-stem DNA probebased electrochemical sensing (5 nM).30 Due to the remarkably high sensitivity of the method, an assay of single nucleotide polymorphism in real samples without pre-enrichment could be achieved. By examining the change in the SPR signals, apoE genotyping and quantification were realized (left panel, Figure 6). Recall that apoE ε2 contains GTGC at codons 112 and D

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biotinylated fragment from the surface hinders the attachment of SA, leading to smaller SPR signals. However, the single-base mismatch in the recognition sites prevents the enzymatic cleavage reaction, and larger SPR signals were attained. The method is amenable to the quantification of the complementary and single-base mismatched apoE sequences, and their concentration levels were determined to be 10 fM and 50 fM, respectively. The genotypes in the assayed four serum samples were deduced to be homozygous apoE ε2/2 and ε3/3 and heterozygous apoE ε2/3 and ε3/4, and the concentration of each genotype was accurately measured. The capability of the method for quantification of apoE gene and genotype discrimination in unamplified genomic DNA extracts provides a viable means for point-of-care diagnosis of neurodegenerative diseases.

Figure 6. Left panel: ApoE genotyping and quantification from 10fold diluted genomic DNA extracts (A, B, C, and D represent the four different DNA samples). The complementary strands with GCGC bases were measured directly through hybridization to the preimmobilized probes, and the strands with a single-base mismatch were quantified by incorporating the complementary strands which competed with the mismatched strands for the preimmobilized probes. All the hybridization reactions were followed by the cleavage reaction and SA attachment. The absolute errors deduced from three replicate measurements are shown as the error bars. Right panel: Genotyping results of the corresponding samples by the signal-base extension assay.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jianxiu Wang: 0000-0002-6344-6419 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

158, apoE ε3 contains GTGC at codon 112 and GCGC at codon 158, and apoE ε4 contains GCGC at codons 112 and 158 (Table 1). For sample A, the existence of GTGC at codons 112 and 158 suggests that the genotype of sample A is apoE ε2/2 (Figure 6A). As for sample B (Figure 6B), the absence of GCGC at codon 112 and the existence of GCGC at codon 158 and GTGC at both codons 112 and 158 indicate that the genotype of sample B is apoE ε2/3. As shown in Figure 6C, because only apoE ε3/3 possesses GTGC at codon 112 and GCGC at codon 158, the genotype in sample C is apoE ε3/3. As depicted in Figure 6D, based on the fact that apoE ε3 possesses GTGC at codon 112 and GCGC at codon 158 and apoE ε4 contains GCGC at codons 112 and 158, the genotype of apoE ε3/4 is present in sample D. Due to the higher risk factor of AD with one or two copies of the ε4 allele,3,5 sample D might be from the patients with suspected AD. Our genotyping results were consistent with those by the single-base extension assay carried out by KingMed Diagnostics Inc. (Tianjin, China; right panel of Figure 6), in which the number of the peaks is indicative of the genotype information. The homozygous apoE ε2/2 in sample A and apoE ε3/3 in sample C both exhibited two peaks, and the heterozygous apoE ε2/3 in sample B and apoE ε3/4 in sample D both possessed three peaks. The base information on codons 112 and 158 presented by the peaks in the single-base extension assay was consistent with the results by the SPR method. However, the single-base extension assay could only provide the genotyping information, and the quantification of the apoE genes could not be achieved. Our method thus serves as a viable means for apoE genotyping and quantification, holding great promise for diagnostic utility for AD.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful for the financial support of this work from the National Natural Science Foundation of China (Nos. 21876208, 21705166, 21575166, 21375150) and the National Key Basic Research Program of China (2014CB744502).

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REFERENCES

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CONCLUSION A proof-of-principle demonstration of apoE genotyping and quantification has been performed on a dual-channel SPR. The restriction enzyme HhaI could selectively cleave the GCGC base pairs in the duplex, and the detachment of the E

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DOI: 10.1021/acssensors.8b00845 ACS Sens. XXXX, XXX, XXX−XXX