Clinically Relevant Detection of Streptococcus pneumoniae with DNA

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Clinically Relevant Detection Of Streptococcus Pneumoniae With DNA-Antibody Nanostructures Jinping Wang, May Ching Leong, Eric Zhewei Leong, Win Sen Kuan, and David T. Leong Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 26 May 2017 Downloaded from http://pubs.acs.org on May 26, 2017

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Analytical Chemistry

Clinically Relevant Detection Of Streptococcus Pneumoniae With DNA-Antibody Nanostructures Jinping Wang,†,# May Ching Leong,†,# Eric Zhe Wei Leong,† Win Sen Kuan,*,‡,§ David Tai Leong*,† †

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore ‡ Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore § Emergency Medicine Department, National University Hospital, National University Health System, 5 Lower Kent Ridge Road, Singapore 119074, Singapore ABSTRACT: Streptococcus pneumoniae (SP) is a pathogenic bacterium and a major cause of community-acquired pneumonia that could be fatal if left untreated. Therefore, rapid and sensitive detection of SP is crucial to enable targeted treatment during SP infections. In this study, DNA tetrahedron (DNA TH) with a hollow structure is anchored on gold electrodes to construct an electrochemical immunosensor for rapid detection of pneumococcal surface protein A (PspA) peptide and SP lysate from synthetic and actual human samples. This DNA nanostructure-based immunosensor displays excellent electrochemical activity towards PspA with a sensitive linear region from 0 to 8 ng/mL of PspA peptide and a low limit of detection (LOD) of 0.218 ng/mL. In addition, this DNA-TH-based immunosensor exhibits good sensing performance towards SP lysate in a clinically relevant linear range from 5 to 100 CFU/mL with a LOD of 0.093 CFU/mL. Along with these attractive features, this electrochemical immunosensor is able to specifically recognize and detect the PspA peptide mixed with other physiologically relevant components like bovine serum albumin (BSA) and lipopolysaccharide. In addition, our sensor could detect SP lysate even when dispersed in BSA or Escherichia coli lysate. Lastly, uncultured samples from the nasal cavity, mouth and axilla of a human subject could be successfully determined by this well-designed electrochemical immunosensor.

Pneumonia is an infection of the lungs that causes the air sacs or alveoli to be filled with fluid or pus, and is often life threatening if left untreated. The World Health Organization estimated just under 1 million deaths from pneumonia in children under 5 years old in 2015, accounting for 16% of all mortality in that age group.1 Pneumonia is caused by a variety of infectious agents, such as bacteria, viruses and fungi. Streptococcus pneumoniae (SP) accounted for 95% of cases of pneumonia in the pre-antibiotic era and in the current age remains the major pathogen implicated in pneumonia currently.2 SP can colonize the upper respiratory tract of healthy carriers. Of concern, in susceptible individuals such as the elderly, children and immunocompromised individuals, SP may spread to other locations including the deep regions of the lung, central nervous system, sinus, middle ear and bloodstream, causing lifethreatening conditions such as pneumonia, meningitis, sinusitis, otitis media, and bacteremia, respectively.3 Previous reports have revealed that there are several humanspecific pathogen-host interactions during SP infection, including the contributions from the metallo-type immunoglobulin A1 protease, choline-binding protein A, enolase and pneumococcal surface protein A (PspA) of SP.4-6 Of importance, this interaction with PspA is pathogenically linked.7 Due to the high morbidity and mortality caused by pneumococcal infection, it is imperative to identify SP infection early via detection of ultralow concentrations of PspA using an ultrasensitive method to achieve rapid diagnosis and guide administration of appropriate antimicrobial therapy in the healthcare setting.

Currently, laboratory diagnosis of SP infection can be performed by polymerase chain reaction (PCR),8 enzyme-linked immunosorbent assay (ELISA),9 antibody tests on blood or urine antigen tests.10,11 PCR is popular as it is very sensitive due to the amplification capabilities but its high sensitivity predisposes this technique to false positives, leading to inappropriate and sometimes dangerous administration of the wrong antibiotics. Moreover, the PCR reaction depends on the purity and quality of the DNA samples, which then necessitates upstream purification steps. Since PCR quantifies DNA, it is challenging to present an accurate clinical diagnosis through PCR assay for the DNA of SP, especially distinguishing between non-pathogenic and pathogenic colonization. Thus, an analyte target that is pathologically linked should be sought. ELISA may also be used to detect PspA. However, the concentration of PspA in body fluids obtained through noninvasive means such as lung condensates is miniscule; below the nanomolar detection range of standard ELISA sensitivity. Electrochemical sensors can fill that gap due to their advantages of simple operation, low cost, high sensitivity and efficiency.12-15 The highly specific recognition between antigens and antibodies also confers high selectivity amongst the diverse microbiome of the upper respiratory tract. Electrochemical immunosensors determine the presence of antigens by detecting the changes in current, capacitance, potential, impedance or conductance resultant from immunoreactions.1622 After the primary immobilized antibody (Ab) captures the antigen (Atg), a secondary enzyme-labelled Ab is injected onto the electrode to form a sandwich assay.23,24 The enzymat-

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ic reaction between the enzyme and enzymatic substrate results in the production of an electroactive molecule, which transmits an electrochemical signal.24 Alternatively, nonenzymatic-labelled-antibody combined electroactive tag could generate large amount of redox markers to achieve electrochemical sensing,25 as shown in Figure 1a. However, the accessibility of targeted objects to the probes on a heterogeneous surface is a major challenge in surface-based biological detection as compared to recognition of probe and target in a homogeneous solution.26 Random projection of antibodies on the surface and high probability of overlapping antibodies could lead to inconsistencies in the capturing of antibodies on the sensory surface. Increasing the concentration of capturing antibodies ironically compounds the problem further as more antibodies tend to overlap over one another without any expected increase in the accessibility of the antigens to the antibodies, as shown in Figure 1b. Recently, DNA nanostructures have garnered tremendous interest in nanotechnology development due to its ability to design conformational predictable self-assembled structures with simple WatsonsCrick base pairing rules.27 Well-designed DNA structures are used in many applications besides its traditional function: genetic information storage.28-30 The DNA nanostructure can be covalently bonded on the surface of a gold electrode via the interaction between thiol groups on DNA strands and gold.31 This process enables the formation of self-assembled monolayer of DNA nanostructures on the surface of gold electrodes, projecting the antibodies in an upright orientation as shown in Figure 1a and b. Moreover, the DNA tetrahedron (DNA TH) has been proven to have mechanical rigidity and structural stability to be used as scaffolds for electrochemical biosensor application.32 The structure can also enhance the overall packing of tetrahedrons by keeping a theoretical spatial separation distance of at least 4 nm, reducing the entanglement between neighboring probes and local overcrowding effect,33,34 as shown in Figure 1b. The hollow structure of the tetrahedron is able to increase the sensitivity of electrochemical detection by enhancing the electron transfer capacity 33 and could also be synthesized rapidly and cost-effectively.34 A recent study reported that DNA TH-based biosensor could selectively detect immunoglobulin G with LOD of 2.8 pg/mL.34 In this study, DNA TH nanostructures were constructed and modified on the surface of gold electrodes, and the PspA antibody was then conjugated with the carboxyl group on the top vertex of the tetrahedron. After incubating BSA on the surface of gold electrode, PspA was utilized as the targeted antigen in the detection of SP. Subsequently, the ferrocene carboxylic acid-conjugated antibody (FeC-Ab) was carefully modified onto the electrode surface to react with PspA. In addition, synthesized PspA peptide was replaced with SP lysate to prove the feasibility of detection of PspA antigen of SP for practical application. This DNA TH-based electrochemical immunosensor performed rapid and sensitive SP sensing ability, obtaining limit of detection as low as 0.218 ng/mL of PspA peptide and 0.093 CFU/mL equivalent of lysed SP. PspA peptide and SP lysate were selectively detected by this electrochemical immunosensor in the presence of various relevant interferences. Moving a step closer to clinical application in this proof-ofconcept study, we tested our system using uncultured SP samples obtained from the upper respiratory tract, mouth and axilla (armpit). Consequently, we conclude that our DNA nanotechnology showed promise as a rapid and sensitive detection tool for SP infections in the healthcare setting.

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MATERIALS AND METHODS Materials and Reagents. PspA peptide and PspA antibody (polyclonal Ab, SC-17481) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Nhydroxysuccinimide (NHS), 1-ethyl-3-(3dimethylaminopropyl), carbodiimide hydrochloride (EDC), bovine serum albumin (BSA), lipopolysaccharides from E. coli O111:B4 (LPS), tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and N-2-hydroxyethylpiperazine-N’-(2ethanesulfonic acid) (HEPES) were purchased from SigmaAldrich (St Louis, MO, USA). Tris base was obtained from 1st Base, Singapore. Tris-Borate-EDTA (TBE) buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3) was obtained from Vivantis Inc. (Oceanside, CA, USA). Sodium dihydrogen phosphate (NaH2PO4), sodium hydrogen phosphate (Na2HPO4), magnesium chloride (MgCl2), and potassium chloride (KCl) were purchased from Merck, Germany. Ferrocene carboxylic acid (FeC-COOH) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All purified oligonucleotides were synthesized by Sangon Biotech (Shanghai) Co., Ltd (China). Strand Name: DNA sequence. TH-A: 5'-HOOCT10 ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A-3'; TH-B: 5'-HS-C6-TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C-3'; TH-C: 5'-HS-C6-TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C-3'; TH-D: 5'-HS-C6-TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T-3'. All solutions were prepared with ultrapure water

(Milli-Q, 18 MΩ·cm resistivity at 25 °C, Merck Millipore, USA). Electrolyte solutions and buffers consisted of phosphate-buffered saline solutions (0.1 M Na2HPO4, 0.1 M NaH2PO4 and 0.1 M KCl, pH 7.4; PBS10x) and its 10-fold diluted solution (PBS1x). Preparation of Ferrocene-conjugated PspA Antibody. The ferrocene-conjugated PspA antibody was fabricated by the method described in previous work.34 Mix 2 mg FeC-COOH with 10 mg NHS and 15 mg EDC in 700 µL HEPES buffer (pH 7.3, 50 mM). After stirring the mixture for 2 hours at room temperature, 300 µL of Ab (10 µg/mL) in PBS1x was added dropwise with continuously stirring for 12 hours to activate the carboxyl groups on FeC-COOH. The mixture was centrifuged twice at 5000 r.p.m for 10 min to remove precipitates (MWCO-10 K, 0.5 mL; Merck Millipore, Billerica, MA, USA). The purified FeC-Ab conjugate was stored at 4 °C. Construction of DNA Tetrahedron-based Immunosensor. The gold electrodes with diameter of 2 mm (CH Instruments, Austin, TX, USA) were polished and electrochemically checked for baseline reading before incubating with the DNA TH. The four DNA strands (TH-A to TH-D) were treated with 30 mM TCEP in 100 mM Tris, 250 mM MgCl2, pH 8.0 buffer to break the disulphide bridges formed between the thiol group between strands during storage. The mixture was heated to 95 °C for 5 min and then cooled to 4 °C for 30 min to form the tetrahedron structure. 3 µL of prepared DNA TH solution was dropped on the freshly cleaned surface of gold electrode and incubated overnight at room temperature, allowing DNA TH to anchor on the gold electrode surface via strong thiol-Au bonds as shown in Figure 1a. This enabled the formation of a self-assembled monolayer of tetrahedra on the surface of gold


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Figure 1. Construction and characterization of DNA tetrahedron and DNA TH-based immunosensor as well as the characterization of FeC-PspA Ab conjugate. (a) Schematic illustration of the construction of DNA-TH-based electrochemical immunosensor. (b) Scheme of antibody immobilization on gold electrode with random orientation and optimized orientation. (c) UV-vis absorption spectra of ferrocene carboxylic acid, PspA antibody, and the conjugate of FeC-Ab. (d) Various combinations of the oligonucleotides were assembled and characterized with non-denaturing polyacrylamide gel electrophoresis. The band of DNA tetrahedrons was shown in lane 8. electrode in a plane normal to the electrode surface. The remaining non-thiolated top vertex was used as a probe for the binding of PspA Ab. Thereafter, 3 µL mixture of EDC (400 mM) and NHS (100 mM) in PBS10x (pH 7.4) was dropped onto the surface of gold electrodes and incubated for 30 min to activate the carboxylic group on the top vertex of DNA nanostructure in order to form amide bond between DNA tetrahedron and Ab. Then, 3 µL of Ab (10 µg/mL) was added onto the gold electrode surface, reacting for 2 hours at room temperature. Next, 1 w/v% BSA solution (in PBS1x) was dropped onto the gold electrode surface and incubated at 4 °C for 30 min to block the non-specific binding sites. Consecutively, 3 µL of various concentrations of antigen was added onto the capture antibody modified electrode (Au-TH-Ab) and incubated at 37 °C for 1 hour to generate the immunoreaction. Finally, 3 µL of FeC-Ab was pipetted onto the surface of the gold electrode and incubated at 37 °C for 1 hour. The entire process is illustrated in Figure 1a. Preparation of Real Samples. Real samples were obtained from the nasal cavity, mouth and axilla using sterile swabs. The swabs were then submerged in 400 µL of Laemmli lysis

buffer and incubated on ice for 30 min. The samples were sonicated at intervals of 20 seconds for 3 times and then used as samples during the immunosensor detection. Electrochemical Measurements. All of the electrochemical measurements were performed on Eco Chemie Autolab PGSTAT 30 electrochemical workstation (Metrohm B. V.). A three-electrode system with a gold working electrode, an Ag/AgCl reference electrode and a platinum wire counter electrode was used in PBS10x throughout the experiments. The measurement range for cyclic voltammetry (CV) was -0.2 V to 0.6 V at a scan rate of 100 mV s-1. In addition, square wave voltammetric (SWV) measurements were carried out over the range of -0.6 V to 0 V.



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Figure 2. CV detection of gold electrodes with various modifications. (a) and (b) CV curves of gold electrodes with different modifications at the scan rate of 100 mV/s in PBS10x solution (pH 7.4). The concentrations of PspA peptide and SP lysate are 5 ng/mL and 103 CFU/mL, respectively. RESULTS AND DISCUSSION Characterization of FeC-PspA-Ab Conjugate. Ferrocene carboxylic acid (FeC) was used as an electroactive tag for electrochemical detection after conjugating with Ab, which can generate electrons during the biosensing detection.33,34 In order to demonstrate that the FeC-Ab has been obtained successfully, UV-vis absorption spectroscopy detection was employed to systematically characterize the FeC, Ab and FeC-Ab conjugate as shown in Figure 1c. FeC-COOH exhibited two obvious absorption peaks at 260 and 301 nm while pure Ab displayed a characteristic protein peak at 280 nm. After conjugation, FeC-Ab conjugates showed an enhanced absorption range with two peaks at 248 and 260 nm along with the disappearance of the peaks of FeC and Ab, indicating the successful conjugation of FeC and PspA Ab. Characterization of DNA Tetrahedron. DNA TH was assembled by A-T and C-G bases complementarity of four single DNA oligonucleotides strands (one strand modified with carboxyl group and three strands modified with thiol group). The construction of DNA TH is illustrated in Figure 1d where the sequences of the same colors in four DNA strands were hybridized and formed the apices of the tetrahedron structure. The DNA TH was validated with 12.5 % polyacrylamide gel electrophoresis as shown in Figure 1d using various combinations of the stranded groups (lacking one, two or three DNA strands) resulting in different relative motifs. Figure 1d also shows that the tetrahedron with a higher molecular weight migrated more slowly than other samples as it consisted of four DNA strands, demonstrating the successful assembly of TH nanostructures.34 Electrochemical Detection of PspA Peptide. Cyclic voltammetry was performed to determine the different modifications on the surfaces of the gold electrodes. As shown in Figure 2, distinctive electrochemical signals were generated from different modified electrodes in PBS10x solution. A pair of oxidation and reduction peaks was obtained at 0.35 V and 0.28 V respectively from the complete assembly of Au-TH-Ab-PspA peptide-FeC-Ab immunosensor, while there was no peak on the bare electrode, TH-modified electrode, TH-Ab-modified electrode and TH-Ab-PspA peptide electrode as shown in Figure 2a. In order to validate the redox peaks were generated on the TH-Ab-PspA peptide-FeC-Ab modified electrode, a control experiment was carried out by replacing the FeC-Ab with

unconjugated Ab. The absence of any observable peak on the electrode modified with Au-TH-Ab-Atg-Ab confirmed that FeC is very important during the electrochemical sensing. Furthermore, there was no observable peak for groups not incubated with PspA peptide as depicted in Figure 2a, demonstrating that there was negligible non-specific binding. Similarly, as shown in Figure 2b, this DNA TH-based immunosensor was used to detect the SP lysate, in which there was only one pair of redox peaks generated on the complete immunosensor at 0.34 V and 0.27 V, respectively. Therefore, the sensor will only register positive voltage responses when the sandwich immunosensor is assembled. SWV was employed to characterize the performance of the electrochemical immunosensor based on the DNA TH. The technique of SWV is able to generate high response signals due to its high sensitivities.35 As the response signals generated in SWV correlate with the concentration of the antigen, various concentrations of antigen were used to study its relationship with the peak current response. Figure 3a shows the typical SWV curve for concentration 8 ng/mL PspA peptide producing a response current of |2.44| µA. As shown in Figure 3b, the complete sandwich immunosensor (DNA TH-Ab-Atg(FeC-Ab)) with 1 ng/mL PspA peptide gave the SWV response current of |0.46| µA, while the sandwich immunoassay without PspA peptide (DNA TH-(FeC-Ab)) showed a low response current of |0.203| µA. In addition, Figure 3b shows the linear relationship i=0.271c+0.203, R2=0.99 where i is magnitude of the peak current (in µA) and c is concentration of PspA (in ng/mL), between the peak current response and the PspA peptide concentration over the range of 0 to 8 ng/mL. A low limit of detection was obtained at 0.218 ng/mL PspA peptide based on a signal-to-noise ratio of 3. We tested the specificity of our system to reduce false positive results in the presence of other non-target proteins of physiological relevance. As shown in Figure 4a, BSA and LPS gave the expected low background signals comparable to the negative control group without any antigen. When the PspA peptide are mixed into various combinations of mixtures containing BSA or LPS or both were tested, the peak current values obtained were similar to the values acquired by the target solely suspended in buffer. The results confirmed that the presence of other molecules did not interfere with the


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Figure 3. SWV detection of PspA peptide and SP lysate. (a) Typical SWV curve for concentration of 8 ng/mL PspA peptide. (b) The peak current responses (absolute values) were plotted versus the corresponding concentration of PspA peptide over the range from 0 to 8 ng/mL. SWV peak current response versus (c) concentration of SP lysate and (d) logarithmic value of SP lysate concentration. Inset: the linear relationship between the current response and the SP lysate with low concentration. The error bars indicate the standard deviations from three independent experiments.

Table 1. Performance comparison of some methods for SP detection. Methods

Linear range [CFU/mL]

Reference

10-106

Limit of Detection (LOD) [CFU/mL] 10

PCR Single tube loop-mediated isothermal amplification assays

100-107

100

37

Real-time PCR

1-107

1

38

Nested-PCR assay

10-106

10

39

Multiplex PCR

100-105

100

40

DNA-TH-based electrochemical immunosensor

5-100

0.093

This work

detection of PspA. The specificity of this detection system demonstrated the powerful advantage of the highly specific recognition between antigen and antibody in detecting antigen which presents in a complex system.41 Therefore, the specificity and selectivity have improved the reliability of this electrochemical sensing system as the possibilities of false positive results.


36


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Figure 4. Specific detection of PspA peptide and SP lysate. (a) The peak current responses of LPS, BSA, and PspA peptide dispersed in PBS1x with 1 % skim milk. (b) The peak current responses of SP lysate, BSA, and E. coli lysate suspended in PBS1x with 1 % skim milk. Data are means ± S.D., n =5, Student’s t-test, p < 0.05, *Significant compared to control. Detecting PspA in SP Lysate. The practical application of this DNA TH based electrochemical sensing system was studied by detecting the presence of PspA in SP lysate due to the successful quantification of synthesized PspA peptide on DNA TH based immunosensor. In Figure 2b, a pair of oxidation and reduction peaks at 0.34 V and 0.27 V was observed only when the sandwich immunosensor was completely constructed with TH-Ab-SP lysate-(FeC-Ab), while there is no observable redox peaks appeared on the electrodes modified with DNA TH, DNA TH-Ab, DNA TH-Ab-(FeC-Ab), DNA TH-Ab-SP lysate, and DNA TH-Ab-SP lysate-Ab. Therefore, it can be concluded that this DNA TH-based detection system could be applied in the detection of SP lysate. The DNA TH based electrochemical immunosensor was utilized to detect SP lysate at different concentrations. As shown in Figure 3c, the peak current responses of SP lysate increased monotonically with increasing concentrations of SP lysate in the range of 0 to 100 CFU/mL. The inset in Figure 3c displays that the peak current response and low concentration of SP lysate (0-20 CFU/mL) have a linear regression correlation of i=0.019x + 0.231 and R2=0.96. Linear correlation (i =0.437 lg c + 0.017; R2=0.99) between the peak current responses of SWV and log of the equivalent concentration of SP lysate was obtained over the range from 5 to 100 CFU/mL of SP lysate (Figure 3d). The LOD was as low as 0.093 CFU/mL of SP lysate based on a signal-tonoise ratio of 3. These analytical parameters have been compared with other reported SP detecting methods (Table 1). Although the linear range is comparatively narrow, within the clinical context of expected ultralow CFU/mL in the patients’ samples, the narrow range is still viable in practice. Moreover, detecting SP in the clinical samples for this detection issue is more important than the quantification of SP as the presence (or absence) of SP would direct the clinician to make a dichotomous course of treatment. In order to demonstrate the specific detection further, this electrochemical immunosensor was challenged to test the SP lysate mixed with Escherichia coli (E. coli) and BSA in skim milk suspension. Similar to the specificity test of PspA pep-

tide shown in Figure 4a, the DNA TH-based immunosensor performed the specificity for SP lysate sensing in the presence of BSA and E. coli lysate as displayed in Figure 4b. Compared to the control group, this immunosensor did not show any significant response towards E. coli and BSA, while SP lysate could be detected and had almost same response in the system with or without E. coli and BSA. Therefore, this DNA-TH-based electrochemical immunosensor could detect SP specifically and selectively and is promising to apply in real sample detection.

Figure 5. The peak current responses and corresponding concentrations of SP in real samples from axilla, nasal cavity and mouth. Error bars refer to standard deviations in 5 independent groups. Real Sample Detection. In order to ensure that the DNA TH-based detection system is capable for clinical application, it is essential to utilize actual samples obtained from the human body where SP are known to be present or absent. Samples from the nasal cavity, mouth, and axilla were obtained from an individual using sterile cotton buds. Figure 5 shows the peak current responses of the human samples and control group. The samples obtained from the axilla showed the same response with the control, demonstrating that there is no SP and correlated to the fact that PspA can hardly be found in the axilla.42,43 However, the samples collected from


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mouth and nose generated much higher responses, having 10 and 6 SPs per group respectively. Even though the samples were obtained from the same individual, the concentration of SP present in the human body may be different from location to location. Therefore, without amplification or long complicated purification steps, this DNA TH-based detection system has proven to detect actual human samples successfully and hence shortening the time required to detect SP as compared to the traditional culture methods. CONCLUSION In this study, the DNA TH-based electrochemical immunosensor has been successfully constructed for SP sensing by detecting PspA on SP membrane. The electrochemical signals generated by ferrocene-labelled sandwich immunosensor were used to quantify the synthesized PspA peptide and SP lysate. The linearity between the absolute peak current responses and the PspA peptide concentration as well as SP lysate concentration was determined in order to quantify SP in real samples. In addition, this DNA TH-based electrochemical immunosensor achieved low limit of detection: 0.218 ng/mL of PspA peptide and 0.093 CFU/mL equivalent of SP lysate, respectively. This immunosensor could selectively detect and quantify SP in the presence of BSA, E. coli, and LPS. Therefore, this specific non-culture immunosensor is able to shorten the diagnosis time without having to separate PspA from non-targeted interferences. Consequently, due to the simple, rapid, sensitive and selective detection system, the presence of SP that causes lifethreatening diseases can be detected convincingly to decrease the morbidity and mortality through timely and accurate treatment for SP infection in pneumonia management.

AUTHOR INFORMATION Corresponding Authors * Email: [email protected] (ORCID: 0000-0001-8539-9062); [email protected]

Author Contributions D.T.L conceived the technology. W.S.K contributed the clinical aspects. J.W., M.C.L and E.Z.W.L performed the experiments. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. # These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge Ministry of Education AcRF (R-297000-414-112) and National University Health System Funding Support (NR13NEM220MP) through the Engineering-Medicine Seed Fund 2013 to D.T.L. and W.S.K. The authors thank Professor En-Tang Kang for allowing access to the electrochemical workstation.

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