Rapid DNA detection using filter paper

Journal Pre-proof Rapid DNA detection using filter paper Yajing Song, Peter Gyarmati

PII:

S1871-6784(18)32003-X

DOI:

https://doi.org/10.1016/j.nbt.2019.10.005

Reference:

NBT 1211

To appear in:

New BIOTECHNOLOGY

Received Date:

19 December 2018

Revised Date:

7 September 2019

Accepted Date:

9 October 2019

Please cite this article as: Song Y, Gyarmati P, Rapid DNA detection using filter paper, New BIOTECHNOLOGY (2019), doi: https://doi.org/10.1016/j.nbt.2019.10.005

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Rapid DNA detection using filter paper Yajing Song*, Peter Gyarmati University of Illinois, College of Medicine, Department of Cancer Biology and Pharmacology, Peoria, IL, USA. * Correspondence E-mail address: [email protected]

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Graphical abstract

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The graphical abstract illustrates the concept of DNA detection using filter paper and brown colored beads. The visual signal of detection can be analyzed quantitatively as the histogram indicates.

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Highlights  Filter paper is suitable for use as a support platform in point-of-care devices. 

Polyamidoamine

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The method can provide a rapid and accurate DNA diagnosis tool.

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

method

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ABSTRACT

Point-of-care (POC) detection is crucial in clinical diagnosis in order to provide timely and specific treatment. Combining polyamidoamine (PAMAM) dendrimer, p-phenylene diisothiocyanate (PDITC) and superparamagnetic beads, a novel method to activate the surface of filter paper to bind DNA molecules has been developed. The method is based on the primary amination of the filter paper surface with PAMAM dendrimer, followed by generation of isothiocyanate groups via PDITC, and subsequent repetition of these two steps. Different parameters of the process have been optimized, including probe printing,

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preparation of target DNAs and detection. The result shows that, due to the highly porous structure of filter paper, high amounts of printed probes, target DNAs and magnetic beads can provide high signal intensities in the detection area via probe/target duplex formation. This method is suitable for rapid, specific and cost-efficient DNA detection on cellulose filter paper. It can be used as a POC device, in particular for diagnosis and treatment management of infectious diseases and identification of antimicrobial drug resistance genes. Abbreviations: POC, point-of-care; PAMAM, polyamidoamine; PDITC, p-phenylene diisothiocyanate; LFA, lateral flow immunoassay; FP, filter paper; DMSO, dimethyl sulfoxide; APTS, 3-aminopropyltriethoxysilane; GA, glutaric anhydride; NHS, N-hydroxysuccinimide; DCC, N,N′-dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide; GND, combination of GA, NHS and DCC.

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Keywords: point-of-care testing, cellulose filter paper, nucleic acid detection, polyamidoamine dendrimer, superparamagnetic beads Introduction

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Early, rapid and accurate diagnostics are critical to clinical management and to prompt and precise patient treatment. Point of care (POC) testing can provide this information at a patient’s location [1-3]. Lateral flow immunoassays (LFAs), the most popular format of POC device, enable rapid detection of a target analyte in urine, blood and other body fluids [4, 5]. Microarray techniques have been developed to improve the throughput of detection, e.g. of DNA detection based on hybridization of probe and target. Diverse support materials for probe/target immobilization have been utilized, such as nitrocellulose and nylon membranes [6-8] or glass [9]. Filter paper (FP) has considerable advantages over traditional DNA hybridization platforms in respect of turnaround time, cost and ease of use. Since it possesses a three-dimensional microstructure, and a pore size larger than that of nitrocellulose [10], FP provides a stronger wicking force and higher surface-to-volume ratio for fluid to transport. Thus, FP appears to be an ideal material for low-cost and easy-to-use POC devices for health care.

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In this report, a novel method to activate the surface of FP to bind DNA by combining principles of LFAs and traditional microarrays is developed, making possible rapid, specific, instrument-free and cost-efficient detection. The aim of this study was to provide a simplified diagnostic platform, which can be applied to POC diagnosis of rapidly progressive diseases. Materials and Methods Materials and reagents Polyamidoamine (PAMAM) dendrimer, generation 4.0 solution (10% in methanol), pphenylene diisothiocyanate (PDITC), dimethyl sulfoxide (DMSO), WHATMAN™ Qualitative filter paper, 3-aminopropyltriethoxysilane (APTS), glutaric anhydride (GA), N-

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hydroxysuccinimide (NHS), N,N′-dicyclohexylcarbodiimide (DCC), N,Ndimethylformamide (DMF), aline-sodium citrate (SSC) buffer and sodium dodecyl sulfate solution (SDS) were from Sigma-Aldrich. PAMAM dendrimer in aqueous solution at 10% solids was from Dendritech. DYNAL MyOne Dynabeads Streptavidin C1 were obtained from Fisher Scientific. A custom-made magnetic stand was used (MagRach16, Germany). DNA Oligonucleotides were purchased from Sigma-Aldrich (Table 1). Comparison of effect of methanol and PAMAM dendrimer to activate FP surface

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The size of paper slides to be activated was 10-12 mm × 15 mm. Eight different methods were compared (Table 2). Method 1, FP-GND: FP was soaked in saturated GA in DMF overnight to produce carboxylic groups on the FP surface; the active ester compounds containing carboxylate was prepared via reaction with 1 M NHS and 1 M DCC in DMF for 4 h. These active ester compounds can be conjugated with amines via amide bonds [11]. Methods 2-4, 1%/3%/10% PAM-GND: the FP surface was aminated with PAMAM dendrimer in methanol for 24 h, followed by production of carboxylic ester groups described in Method 1. Methods 5-7, GND-1%/3%/10% PAM-GND: The carboxylic ester groups were generated first via reaction between the saturated GA and 1 M NHS/DCC in DMF on the FP surface, followed by the process of Methods 2-4. Method 8, GND-methanol-GND: Methanol was used in place of PAMAM in Methods 5-7. Untreated FP was used as a negative control. All the above steps were completed at RT.

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Further comparison of active effect of APTS and PAMAM during combinations of PDITC, GA-DCC-NHS or PDITC / GA-DCC-NHS

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The functionalization effect was compared based on ten different combinations of APTS, PAMAM, PDITC, GA, DCC/NHS (Table 3).

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APTS-GND: Amino groups (-NH2) were produced through the reaction between APTS (volume ratio of APTS: H2O: ethanol = 2:3:95) [12] and hydroxyl groups (-OH) of FP for 24 h. The APTS solution was added to the FP slides. Amine-reactive ester compounds on the FP surface were prepared by coating with saturated GA in DMF overnight followed by reaction with 1 M NHS/DCC in DMF for 4h at RT. APTS-GND-APTS-GND: the steps of APTS-GND were repeated on the same FP. APTS-PDI: FP slides were immersed in 10 μM PDITC in DMSO after APTS overnight incubation to create isothiocyanate groups on the surface of FP. APTS-PDI-APTS-PDI: APTS-PDI was repeated on the same FP. APTS-PDI-APTS-GND: the last step of APTS-PDI-APTS-PDI was modified by GND to prepare active ester groups. 100 μL of PAMAM dendrimer (10% in methanol) replaced APTS in all the above five combinations. Thus, the following combinations were also evaluated, respectively: PAMGND, PAM-GND-PAM-GND, PAM-PDI, PAM-PDI-PAM-PDI and PAM-PDI-PAMGND. 3

The operational guideline was to immerse the entire paper slide into the solution containing the APTS or dendrimer. Washing and drying After reactions with GA in DMF and NHS/DCC in DMF, the FP slides were washed by DMF in both of the steps. After the steps, either with PAMAM in methanol or methanol alone, methanol itself was applied to wash the paper slides. Ethanol was used to wash FP after generation of amino groups from APTS on the surface of FP. DMSO was used in a wash step after activation with isothiocyanate groups from PDITC in DMSO. All wash steps were for 5 min followed by a water wash for 3 min after which FP slides were dried. The entire functionalization procedure was completed at RT.

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Probes and target

Probe immobilization

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The probes (APT2, TID, and APT2WO) and target (RE-APT2) were synthetic oligonucleotides. Probe structure included amino group modification, 6 or 12 carbon spacer, polythymine (15dT) spacer, and the main body of a sequence from 5’ to 3’ end (Table1) [13]. APT2 [14] was the probe complementary to the sequence of target. TID [13] and APT2WO were two negative control probes for identification of detection specificity. The main sequences of APT2WO and APT2 were identical except for the modification at the 5’ end of oligonucleotides. The three probes were immobilized on each activated FP surface. RE-APT2 was biotinylated at the 5’ terminal and used to detect the probe of aminated-APT2 printed on the activated FP surface.

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The size of each FP slide was 10-12 mm x 15 mm. There were four areas for probe immobilization (Figure 1A). 1.5 µL of each 20 μM probe was printed manually in positions 0 (APT2), 2 (TID) and 3 (APT2WO). The probes were dissolved in printing buffer (50mM sodium phosphate buffer at pH 8-9) (PBF), and PBF was printed in position 1 as a blank control. The slides were incubated for 24 h in a humid chamber at RT, then the untreated active groups of PDITC were blocked with a solution of 50 mM ethanolamine, 100 mM Tris, pH 9.0 at 55°C for 30 min in order to avoid unspecific signals during detection. TID and APT2 WO were two negative controls. This was followed by washing with 4× SSC buffer with 0.1% SDS for 30 min at 55°C and then the printed FP slides were dried at RT. Preparation of ssDNA bound magnetic beads and detection Streptavidin-coated paramagnetic beads were used to label biotinylated target ssDNAs (REAPT2). An amount of RE-APT2 (e.g. 30 pmol) in bind/wash buffer (0.01 M Tris-HCl, 1 mM EDTA, 2 M NaCl, 1 mM β–Mercaptoethanol, 0.1% Tween 20, pH 7.5) [13] was incubated with a volume of paramagnetic beads (e.g. 6 μL) in a rotator for 10 min at RT. The suspension was removed after the tube was incubated on a magnetic stand for 2 minutes. The target ssDNAs labeled with magnetic beads were then washed with 200 μL PBS-T 4

twice and dissolved in PBS-T for detection. For each paper slide detection, 50 μL of target in PBS-T was required. Detection was performed twice on each paper slide (25 μL × 2). The printed paper slide was vertically touched into 25 μL of RE-APT2 labeled magnetic beads solution in PBS-T for about 2 min. The FP was then washed with 2 × SSC buffer with 0.1% SDS at 55° C for 10 min, following a wash with 0.2 × SSC buffer for 1 min at RT, a wash with water for 1 min, and finally dried at RT. The repeated detection was carried out with the remaining 25 μL of RE-APT2-beads solution on the same paper slide as described above. Triplicate detections were performed under each condition. Image and statistical analysis

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The myImageAnalysis v1.1 software was used to measure signal intensities (Fisher Scientific). The signal intensity from each printed area was measured based on the natural brown color of the iron-oxide magnetic beads. The brown color was visible by the naked eye in contrast to the white of FP and the image was captured to quantify signal intensities. The intensities of “APT2-PBF” (subtracting the intensity of position 1 from that of position 0) were analyzed and compared between the different methods. Student’s t-test was used to estimate significance, with p ≤ 0.05 as significance level. Optimization of parameters in the selected method

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Based on the method of PAMAM-PDITC-PAMAM-PDITC, three concentrations of PDITC (10 mM, 20 mM and 30 mM), two different volumes (80 μL and 100 μL) of PAMAM dendrimer in methanol and two kinds of carbon spacer (carbon 6 and carbon 12) were used in optimization. 30 pmol of target ssDNAs were bound to 6 μL of magnetic beads and detected 1.5 μL of printed probes (20 μM). The signal intensities of “APT2-PBF” were calculated and compared under different conditions listed above. Comparison of activation effect on FP surface with PAMAM either in methanol or in deionized water

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In order to investigate the efficacy of functionalization with PAMAM dendrimer in methanol (pH > 9) and in deionized water (pH = 8.5 and pH = 4.6), 100 μL of PAMAM dendrimer in methanol and 150 μL of PAMAM dendrimer in deionized water were used with 20 μM of PDITC in DMSO and APT2 probes modified with C12 at the 5’ end. 1.5 μL of printed probe (20 μM), 3 μL of magnetic beads, and 1.5 μL of target oligonucleotide (10 μM) were applied to complete printing and detection. The signal intensities of “APT2-PBF” subtracted from active FP were analyzed and compared. The activated FP slides were aminated with 10% PAMAM in methanol or in deionized water (wt% solids). Paper slide size was 12 mm × 15 mm. Optimization of amounts of immobilized probes and target oligonucleotides and volume of magnetic beads

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To optimize the amount of immobilized probes, three different volumes of each probe (1.5 μL, 3 μL and 6 μL) were investigated sequentially in the same printed layout. PBF was used as a blank control. 3 μL of magnetic beads and 1.5 μL of target oligonucleotides (10 μM) were used for detection. In order to investigate the amount of target ssDNAs, 30 pmol and 60 pmol of target ssDNAs were used to detect 6 μL of printed probes (20 μM) on the active FP, combining 3 μL of beads in each condition. Later, three distinct volumes of beads (3 μL, 4.5 μL and 6 μL) were compared by identifying 6 μL of printed probes bound with C12 (20 μM) on the activated surface of FP. 60 pmol of target nucleic acid were used in the detections. The signal intensities of “APT2-PBF” were compared in all optimizations. Evaluation of detection limit based on synthetic oligonucleotides

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In order to investigate limit of detection of the current system, different amounts of target RE-APT2 (0, 0.1, 10, 60, 100, 120 pmol) were used to detect 120 pmol of immobilized probe oligonucleotide on activated FP with 6 μL magnetic beads. The reactions were performed in triplicate under each condition. Evaluation of specificity of detection in the developed system

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In order to evaluate detection specificity, synthetic oligonucleotides containing random mutations were used (mutation rates: 0%, 5%, 10%, 25% and 50%). Mutated bases were selected using random.org, a true random number generator. 120 pmol of printed probes, 60 pmol of target ssDNAs and 6 μL of magnetic beads were used for each active FP detection. The reactions were performed in triplicate under each condition. Results and Discussion

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Specific signals were only observed in the area of target probe (APT2 in position 0) instead of the areas of controls (PBF in position 1, TID in position 2, and APT2WO in position 3). The background was mainly generated in the position 1 area (Figure 1B). Therefore, the signal intensities of “APT2-PBF” subtracted from active FP were calculated and compared.

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Evaluation of the efficiency of surface activation systems on cellulose filter paper

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The generation 4.0 PAMAM dendrimer contains 64 primary surface amino groups (-NH2), which can attach covalently to the surface of glass to maximize the printed probe [12]. There is potential for PAMAM dendrimer to increase the surface area of cellulose FP based on its unique porous structure compared with glass or nitrocellulose, so as to increase density and homogeneity of signal intensities. Thus, PAMAM in methanol, in different concentrations, was investigated. Based on GA, NHS/DCC, PAMAM dendrimer and methanol, eight different methods were investigated (Figure 2A, B) at different concentrations of PAMAM dendrimer. There was a positive correlation between concentrations of PAMAM and signal intensities using PAMAM-GND, but negatively correlated with that of GND-PAMAM-GND (Figure 2A). 6

The method with 10% PAMAM-GND displayed the highest intensity compared with the signals in the other 7 methods (Figure 2A). The difference between 10% PAMAM-GND and GND-Methanol-GND was significant (p = 0.008). APTS is commonly used in derivatizing an active surface of glass slides [15]. It was reported that PDITC possessed homobifunctional isothiocyanate groups, which formed a polymeric network by reacting with multiple amino groups of PAMAM [12]. Application of GA, DCC, and NHS to activate glass slides step-by-step avoided the formation of a crosslink in one step and provided a higher signal compared with the method containing PDITC [12].

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Ten methods were explored to evaluate the activation effect with APTS or 10% PAMAM dendrimer in methanol (wt% solid) with combinations of GA-NHS-DCC or PDITC or PDITC together with GA-NHS-DCC. The use of PAMAM-GND with 10% dendrimer was compared with the other nine methods. Non-treated FP was used as a negative control (Figure 2C). The result presents the signal intensities of different methods with PAMAM being significantly higher than that with APTS (p = 0.03). The strongest signal intensity was obtained from treatment with PAMAM-PDITC-PAMAM-PDITC (Figures 1B and 2C). There was a significant difference between the signal intensities of the PAMAM-GND and PAMAM-PDITC-PAMAM-PDITC methods (p = 0.001, Figure 2C).

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Evaluation of parameters in the PAMAM-PDITC-PAMAM-PDITC method

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The effect was investigated of 10mM, 20mM and 30mM PDITC based on the PAMAMPDITC-PAMAM-PDITC combination and the signal intensities analyzed by subtracting the volume of PBF from that of APT2. The strongest signal was detected when using 30mM PDITC and the increase in concentration resulted in a linear increase in signal intensity (Figure 3A, R2 = 0.99). A significant difference was observed between the signal intensities for 10mM and 30mM PDITC (p = 0.02), while there was no significant difference between the signal intensities of 20mM and 30mM PDITC (p = 0.06). Combining the differences in detection of signal intensities, visual inspection and cost of the assay, 20mM PDITC was chosen for further work. The investigation of the volume of PAMAM dendrimer in this method illustrates that activation with 100 μL of dendrimer produced a stronger signal intensity than that of 80 μL PAMAM dendrimer in methanol (Figure 3B, p = 0.003).

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Due to the 3D structure of FP, 1μM size of beads and electrostatic repulsion between the negatively charged probe and target ssDNAs, the distance of spacer arm between tethered probes and surface of FP is necessary for complementary oligonucleotides to hybridize efficiently on the active surface of FP. The spacer has two major functions. First, the maximum probe yield can be immobilized on the FP surface. Secondly, the target ssDNA can be kept at a distance from the FP surface to reduce steric hindrance during hybridization [16]. Thus, the 12-carbon spacer significantly increased the signal intensities of targets after hybridization compared to the 6-carbon spacer (Figure 3C, p = 0.02). It is also possible

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to further extend spacer length to enhance the detection sensitivity/efficiency of hybridization [10,14,15].

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PAMAM dendrimers are hydrophilic. The activation effect was compared with PAMAM dendrimers in methanol (pH > 9) and in deionized water (pH < 9), which showed that the signal intensity in water was stronger than that in methanol. Further investigation of the signal intensities from PAMAM dendrimers in aqueous solution at different pH values showed that, at pH 4.6, intensity was higher than at pH 8.5, although the difference was non-significant (p = 0.27) (Figure 3D). A possible explanation is that the size of PAMAM dendrimer is altered at different pH values due to electrostatic repulsion between protonated amines, implying that the size of the dendrimer at lower pH value is larger than at higher pH, and molecular accessibility to amines is therefore increased [17]. Thus, PAMAM dendrimer in aqueous pH 8.5 was selected for follow-up work as it generated less background than at pH 4.6, based on visual observation. Optimization of amounts of printed probe, target DNA and volume of magnetic beads

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Once the method for activation of the FP surface was selected, the parameters of detection were optimized. It has been reported that the printed probe density is negatively correlated with hybridization efficiency due to electrostatic repulsion on a planar surface [16]. Our results show that there is a potential to use even more probes, targets and beads for higher signal intensities. In this study, 120 pmol of printed probes, 60 pmol of target ssDNAs and 6 μL of magnetic beads were applied and resulted in the strongest signals, respectively (Figure 4A-C). The reason is that the filter paper used can provide a larger surface space than a planar platform. Moreover, this space is distributed into three dimensions with curved surfaces from FP fibers, which helps to reduce electrostatic interaction between probes during immobilization, and steric hindrance between probes and targets during hybridization. The relaxation of electronic repulsion allows a higher density of aminated probes to be printed on the active surface and more target DNAs can be used for duplex formation. The superparamagnetic beads are coated with a monolayer surface of streptavidin. Due to the high affinity of the streptavidin-biotin interaction, biotinylated target ssDNAs can bind beads and be collected magnetically, and further carry out the specific detection of biotinylated target nucleic acid. The combination of 3D tethered probes on the FP surface with 3D targets on the bead surfaces increases the opportunity for duplex formation. In the current system, the detection signals generated from 0.1 pmol of RE-APT2 were identified by the naked eye although there was a non-significant difference between the intensities from 0.1 and 10 pmol of RE-APT2 (Figure 5A). Thus, the detection limit was about 0.1 pmol per assay. Detection specificity of target (RE-APT2) showed a linear increase in signal intensities with decreased mutation rates of the target sequences (Figure 5B). In this study, the controls of APT2WO and TID qualitatively illustrated that the background noise originated from either the same sequence as probe, but without modification of amino groups, or the distinct probe sequence, but with amino group modification. In all cases,

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neither printed area showed printed spots except for the activated condition with PAMAM dendrimer in deionized water (pH 4.6). A stricter wash condition would be required to increase the signal/noise ratio after the step of amination with PAMAM dendrimer in water (pH 4.6).

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Here, several methods to activate the surface of FP have been explored. Considering the particular structure of FP and hybridization efficiency at RT, possible parameters were further optimized to increase sensitivity and specificity of the assay. The cost of the method developed mainly comes from printed probes, target ssDNAs, magnetic beads and FP. The total cost per assay was approximately $1.00 (15 pmol of printed probes, 15 pmol of target oligonucleotides, 3 μL of magnetic beads and one 12 mm × 15 mm FP slide) to $2.50 (60 pmol probes, 60 pmol target oligonucleotides, 6 μL of magnetic beads and one 12 mm × 15 mm FP slide). Probe immobilization was performed manually in the current study. These features enable the application of FP detection in any geographical distribution as printing and detection do not require instrumentation. However, robotic printing can increase the density of immobilized probes, with decreased printed area, so that both sensitivity and specificity in detection can be increased [10,14]. In this study, 1 μM diameter beads were used. Bead sizes will be evaluated since smaller beads provide a more curved surface than larger ones in a given surface space, achieving a higher target-loading capacity [16].

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Conclusions

Funding

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PAMAM dendrimers can extend the surface area of FP due to its 3D microstructure. Here, a new method was developed in which PAMAM dendrimer and PDITC were applied to activate FP. All the methods and techniques along with the chemicals are publicly available compared to [10, 13]. This work, combined with potential improvements in sensitivity and specificity, provides a potential POC tool for rapid DNA detection (e.g. DNA of pathogens and antimicrobial resistance genes). It takes around 30 minutes from preparing detection solution to completing detection.

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This work was supported by a startup fund from the University of Illinois College of Medicine at Peoria, and a research grant from the Rising Tide Foundation (CCR-17-400).

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Author Contributions

YS and PG contributed the idea and designed the experiments. YS performed the experiments, the analysis of images and data and drafted the manuscript. PG revised the manuscript and provided reagents. Disclosure The authors declared no competing financial interest.

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References

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[1] St-Louis P. Status of point-of-care testing: promise, realities, and possibilities. Clin Biochem. 2000; 33: 427-40. [2] Tsalik EL, Bonomo RA, Fowler VG Jr. New molecular diagnostic approaches to bacterial infections and antibacterial resistance. Annu Rev Med. 2018; 69: 379-94. doi: 10.1146/annurev-med-052716-030320. [3] Dincer C, Bruch R, Kling A, Dittrich PS, Urban GA. Multiplexed point-of-care testing – xPOCT. Trends Biotechnol. 2017; 35: 728-42. doi: 10.1016/j.tibtech.2017. 03. 013. [4] Chen A, Yang S. Replacing antibodies with aptamers in lateral flow immunoassay. Biosens Bioelectron. 2015; 71: 230-42. doi: 10.1016/j.bios.2015. 04. 041. [5] Mohd Hanafiah K, Arifin N, Bustami Y, Noordin R, Garcia M, Anderson D. Development of multiplexed infectious disease lateral flow assays: Challenges and Opportunities. Diagnostics (Basel). 2017; 7: pii: E51. doi: 10. 3390/diagnostics7030051. [6] Conner BJ, Reyes AA, Morin C, Itakura K, Teplitz RL, Wallace RB. Detection of sickle cell beta S-globin allele by hybridization with synthetic oligonucleotides. Proc Natl Acad Sci U S A. 1983; 80: 278-82. [7] Meinkoth J, Wahl G. Hybridization of nucleic acids immobilized on solid support. Anal Biochem. 1984; 138: 267-84. [8] Saiki RK, Walsh PS, Levenson CH, Erlich HA. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A. 1989; 86: 6230-4. [9] Masko U, Sothern EM. Oligonucleotide hybridizations on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotides synthesised in situ. Nucleic Acids Res. 1992; 20: 1679-84. [10] Song Y, Gyarmati P, Araújo AC, Lundeberg J, Brumer H 3rd, Ståhl PL. Visual detection of DNA on paper chips. Anal Chem. 2014; 86:1575-82. doi: 10. 1021/ac403196b. [11] Staros JV. N-hydroxysulfosuccinimide active esters: bis (N-hydroxysulfosuddinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein crosslinkers. Biochemistry. 1982; 21: 3950-5. [12] Benters R, Niemeyer CM, Drutschmann D, Blohm D, Wöhrle D. DNA microarrays with PAMAM dendritic linker systems. Nucleic Acids Res. 2002; 30: E10. [13] Araújo AC, Song Y, Lundeberg J, Ståhl PL, Brumer H 3rd. Activated paper surfaces for the rapid hybridization of DNA through capillary transport. Anal Chem. 2012; 84: 33117. doi: 10. 1021/ac300025v. [14] Chumphukam O. Proximity dependent ligation selection: a new approach to generating DNA aptamers. Dept. of Chemistry. Imperial College, London SW7 2AZ, UK. 2013. https://spiral.imperial.ac.uk:8443/handle/10044/1/24836. [15] Guo Z, Guilfoyle RA, Thiel AJ, Wang R, Smith LM. Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Res. 1994; 22: 5456-65. [16] Ravan H, Kashanian S, Sanadgol N, Badoei-Dalfard A, Karami Z. Strategies for optimizing DNA hybridization on surfaces. Anal Biochem. 2014; 444: 41-6. [17] Maiti PK, Cagin T, Lin ST, Goddard WA 3rd. Effect of solvent and pH on the structure of PAMAM dendrimers. Macromolecules. 2005; 38: 979-91.

Figure 1. Layout of printing locations and comparison of visual signals. A: The detected probes were printed in position 0. In position 1, 1× PBF was located. Two negative controls

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(TID and APT2WO) were printed in position 2 and 3, respectively. B: Comparison of three activated methods. PAMAM-PDITC-PAMAM-PDITC method displays the clearest and highest intensity. All the work was triplicate.

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Figure 2. Overview of the functionalized methods. A: Investigation of eight activated methods on the surface of filter paper (FP). FP is a blank control. GND-Methanol-GND is a positive control. B: There is a significant difference between the signal intensities of GND-Methanol-GND and 10%PAM-GND. C: Evaluation of ten methods using APTS or PAMAM with combinations of GND or PDITC or GND and PDITC. PAM-GND is a positive control. For detailed description see Methods section.

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Figure 3. Parameter optimization in the method using PAMAM-PDITC-PAMAM-PDITC. A: Comparison of PDITC concentration. B: Comparison of volume of PAMAM in methanol. C: Carbon spacer arm comparison. D: Comparison of “APT2-PBF” intensities from different solution containing PAMAM dendrimer.

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Figure 4. Optimization of the amounts of printed probes (A: 20 μM), target DNAs (B) and magnetic beads volume (C).

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Figure 5. Illustration of detection limit (A) and detection specificity (B). Dash-dotted line displays the limit of detection.

Table 1 The sequences and their modifications of probes and targets in this study. 5’ terminal Carbon modification spacer

Polythymine spacer

Main sequences

12

Amino group

APT2WO

None

C6 C12 C6

or

PolyT (15)

TID

Amino group

C6

RE-APT2

Biotin

None

None

5% mutation of RE-APT2 10% mutation of RE-APT2 25% mutation of RE-APT2

Biotin

None

None

Biotin

None

None

Biotin

None

None

50% mutation of RE-APT2

Biotin

None

None

PolyT (15) PolyT (15)

CGCATACCTCTCCAATCTCCG TTTACTGCACCTAATCACCT CGCATACCTCTCCAATCTCCG TTTACTGCACCTAATCACCT AAATTTGCCGACTCGCATAGG TCTGTGATA AGGTGATTAGGTGCAGTAAAC GGAGATTGGAGAGGTATGCG ACGTGATTAGGTGCAGTAAA CGGAGTTGGAGAGGTATGCG ACGTGATTAGGTGCAGTAAA CGGAGTTTGGTGAGGTATCCG ACGTGTTTTGGAGC GTAATCCCAG TTTGGTGAGGTATCCG ACGTGTTTTCGAGGACTAAT GCCACTAAGCTGACGTATCCC

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APT2

Table 2 Effect Comparison of methanol and PAMAM dendrimer to activate the surface of filter paper. A detailed description of the methods can be found in the main text. Step 2

FP-GND

GA in DMF

(2)

1%PAM-GND

(3)

3%PAM-GND

(4)

10%PAM-GND

(5)

GND-1% PAMGND GND-3% PAMGND GND-10% PAM-GND GNDmethanol-GND

1%PAMAM in methanol 3%PAMAM in methanol 10%PAMAM in methanol GA in DMF

(8)

GA in DMF NHS/DCC in DMF NHS/DCC in DMF NHS/DCC in DMF NHS/DCC in DMF

lP

(7)

GA in DMF

GA in DMF

GA in DMF GA in DMF

na

(6)

Step 3

NHS/DCC in DMF GA in DMF

Step 4

NHS/DCC in DMF NHS/DCC in DMF NHS/DCC in DMF 1%PAMAM in methanol 3%PAMAM in methanol 10%PAMAM in methanol methanol

Step 5

-p

Step 1

(1)

re

Method

GA in DMF GA in DMF GA in DMF GA in DMF

NHS/DCC in DMF NHS/DCC in DMF NHS/DCC in DMF NHS/DCC in DMF

Table 3 Comparison of active effect of APTS and PAMAM to activate the surface of filter paper. A detailed description of the methods can be found in the main text. (1) (2)

Step 2

Step 3

APTS

GA in DMF

APTS-GNDAPTS-GND APTS-PDI

APTS

GA in DMF

NHS/DCC in DMF NHS/DCC in DMF

APTS

APTS-PDIAPTS-PDI APTS-PDIAPTS-GND PAM-GND

APTS

PAMAM

PDITC in DMSO PDITC in DMSO PDITC in DMSO GA in DMF

PAM-GNDPAM-GND PAM-PDI

PAMAM

GA in DMF

PAMAM

PDITC DMSO

Jo

(3)

Step 1

APTS-GND

ur

Method

(4) (5) (6) (7) (8)

APTS

APTS APTS NHS/DCC in DMF NHS/DCC in DMF

Step 4

Step 5

Step 6

APTS

GA in DMF

NHS/DCC in DMF

PDITC in DMSO GA in DMF

PAMAM

NHS/DCC in DMF GA in DMF

NHS/DCC in DMF

in

13

(9)

PAMAM PAMAM

PDITC DMSO PDITC DMSO

in

PAMAM

in

PAMAM

PDITC in DMSO GA in DMF

NHS/DCC in DMF

Jo

ur

na

lP

re

-p

ro of

(10)

PAM-PDIPAM-PDI PAM-PDIPAM-GND

14