Harnessing aptamers for electrochemical detection of endotoxin

doi:10.1016/j.ab.2012.02.016

Analytical Biochemistry

Volume 424, Issue 1, 1 May 2012, Pages 12-20

https://doi.org/10.1016/j.ab.2012.02.016 Get rights and content

Abstract

Lipopolysaccharide (LPS), also known as endotoxin, triggers a fatal septic shock; therefore, fast and accurate detection of LPS from a complex milieu is of primary importance. Several LPS affinity binders have been reported so far but few of them have proved their efficacy in developing electrochemical sensors capable of selectively detecting LPS from crude biological liquors. In this study, we identified 10 different single-stranded DNA aptamers showing specific affinity to LPS with dissociation constants (K_d) in the nanomolar range using a NECEEM-based non-SELEX method. Based on the sequence and secondary structure analysis of the LPS binding aptamers, an aptamer exhibiting the highest affinity to LPS (i.e., B2) was selected to construct an impedance biosensor on a gold surface. The developed electrochemical aptasensor showed excellent sensitivity and specificity in the linear detection range from 0.01 to 1 ng/mL of LPS with significantly reduced detection time compared with the traditional Limulus amoebocyte lysate (LAL) assay.

Section snippets

Materials

LPS from E. coli 055:B5 (L4524) was purchased from Sigma-Aldrich (USA). Bare fused-silica capillaries (TSP075375) with a length of 80 cm and inner and outer diameters of 75 and 363 μm, respectively, were purchased from Polymicro (Polymicro Technologies, USA). d-(+)-Glucose (G7021), sucrose (84097), cholesterol (C3045), Baker’s yeast RNA (R6750), and bovine serum albumin (BSA, A7906) were from Sigma (St. Louis, MO, USA). Baker’s yeast RNA was further purified using LPS removal solution (Sigma,

Selection of LPS affinity aptamers

In order to screen aptamers showing high affinity to LPS using NECEEM, it is important to define a collection window where the LPS–aptamer complex is likely to be populated. For this, migration times of free LPS and free ssDNA library were determined under the CE running condition. Since LPS gave no apparent UV or fluorescence signal, its migration time was estimated by collecting LPS in six fractions (from 0 to 48 min, 8 min/fraction) and quantifying LPS in each fraction by LAL assay. It was

Conclusions

A high affinity LPS binder B2 was identified by a NECEEM-based non-SELEX method. To explore the possibility of harnessing B2 aptamer as a specific sensor probe to detect LPS in solution, an electrochemical apatasensor based on aptamer/MCH-mixed SAM on the gold electrode was fabricated. The developed aptasensor was demonstrated to detect LPS at a range of 0.01–1 ng/mL (i.e., at a femtomolar level comparable to the conventional LAL assay) in 15 min. Furthermore, the aptasensor showed little

Acknowledgments

This work was supported by research Grants (NRF-C1AAA001–2011-0018903) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (MEST), Korea.

References (40)

Y.G. Kim et al.
Screening of LPS-specific peptides from a phage display library using epoxy beads
Biochem. Biophys. Res. Commun.
(2005)
A.C. Issekutz
Removal of gram-negative endotoxin from solutions by affinity chromatography
J. Immunol. Methods
(1983)
S. Minobe et al.
Characteristics of immobilized histamine for pyrogen adsorption
J. Chromatogr. A
(1983)
F.B. Anspach et al.
Removal of endotoxins by affinity sorbents
J. Chromatogr. A
(1995)
T.E. Karplus et al.
A new method for reduction of endotoxin contaminations from protein solutions
J. Immunol. Methods
(1987)
P.M. Montbriand et al.
Improved method for the removal of endotoxin from DNA
J. Biotechnol.
(1996)
B. Vant-Hull et al.
The mathematics of SELEX against complex targets
J. Mol. Biol.
(1998)
A. Wen et al.
A novel lipopolysaccharide-antagonizing aptamer protects mice against endotoxemia
Biochem. Biophys. Res. Commun.
(2009)
H. Cai et al.
Label-free protein recognition using an aptamer-based impedance measurement assay
Sens. Actuators B
(2006)
A. Merkoci et al.
New materials for electrochemical sensing VI: carbon nanotubes
TrAC Trends Anal. Chem.
(2005)

M.K. Patel et al.

Electrochemical DNA sensor for Neisseria meningitidis detection

Biosens. Bioelectron.

(2010)

J.Y. Heras et al.

Electronic tongue for simultaneous detection of endotoxins and other contaminants of microbiological origin

Biosens. Bioelectron.

(2010)

J.-D. Qiu et al.

A label-free amperometric immunosensor based on biocompatible conductive redox chitosan-ferrocene/gold nanoparticles matrix

Biosens. Bioelectron.

(2009)

M.J. Shin et al.

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification

J. Chromatogr. A

(2011)

W. Lee et al.

Immobilization of antibody fragment for immunosensor application based on surface plasmon resonance

Colloids Surf. B

(2005)

W.Q. Su et al.

Synthesis, characterization and self-assembled film of poly(3-((2,3-dihydrothieno[3,4-b][1,4]dioxin-2-yl)methoxy)propane-1-thiol) (PEDTMSHA)

Synth. Met.

(2010)

U.K. Sur et al.

Cyclic voltammetric and electrochemical impedance studies on the structure, adsorption kinetics, and barrier properties of some organic dithiol self-assembled monolayers on gold

J. Colloid Interface Sci.

(2003)

F. Lucarelli

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

Biosens. Bioelectron.

(2004)

A. Preston et al.

The lipooligosaccharides of pathogenic gram-negative bacteria

Crit. Rev. Microbiol.

(1996)

R.J. Ulevitch et al.

Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin

Annu. Rev. Immunol.

(1995)

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This work describes fabrication of gold electrodes modified with peptide conjugate DAL-PEG-DK5-PEG-OH that enables ultra-sensitive detection of lipopolysaccharide (LPS) isolated from the reference strain of Escherichia coli O26:B6. The initial step of the established procedure implies immobilization of the fully protected DAL-PEG-DK5-PEG-OH peptide on the surface of the gold electrode previously modified by cysteamine. Then side chain- and Fmoc-deprotection was performed in situ on the electrode surface, followed by its incubation in 1 % of BSA solution to block non-specific bindings sites before LPS detection. The efficiency of the modification was confirmed by X-ray Photoelectron Spectroscopy (XPS) measurements. Additionally, the cyclic voltammetry (CV) and electrochemical impendance spectroscopy (EIS) were employed to monitor the effectiveness of each step of the modification. The obtained results confirmed that the presence of the surface-attached covalently bound peptide DAL-PEG-DK5-PEG-OH enables LPS detection by means of CV technique within the range from 5 × 10⁻¹³ to 5 × 10⁻⁴ g/mL in PBS solution. The established limit of detection (LOD) for EIS measurements was 4.93 × 10⁻²¹ g/mL with wide linear detection range from 5 × 10⁻²¹ to 5 × 10⁻¹⁴ g/mL in PBS solution. Furthermore, we confirmed the ability of the electrode to detect LPS in a complex biological samples, like mouse urine and human serum. The effectiveness of the electrodes in identifying LPS in both urine and serum matrices was confirmed for samples containing LPS at both 2.5 × 10⁻¹⁵ g/mL and 2.5 × 10⁻⁹ g/mL.
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In this study, an ultrasensitive electrochemiluminescence (ECL) aptasensing method was developed for lipopolysaccharide (LPS) determination based on CRISPR-Cas12a accessory cleavage activity. Tris (2,2'-bipyridine) dichlororuthenium (II) (Ru(bpy)₃²⁺) was adsorbed on the surface of a glassy carbon electrode (GCE) coated with a mixture of gold nanoparticles (AuNPs) and Nafion film via electrostatic interaction. The obtained ECL platform (Ru(bpy)₃²⁺/AuNP/Nafion/GCE) exhibited strong ECL emission. Thiol-functionalized single-stranded DNA (ssDNA) was modified with a ferrocenyl (Fc) group and autonomously assembled on the ECL platform of Ru(bpy)₃²⁺/AuNP/Nafion/GCE via thiol-gold bonding, resulting in the quenching of ECL emission. After hybridization of the LPS aptamer strand (AS) with its partial complementary strand (CS), the formed double-stranded DNA (dsDNA) could activate CRISPR-Cas12a to indiscriminately cleave ssDNA-Fc on the surface of Ru(bpy)₃²⁺/AuNP/Nafion/GCE, resulting in recovery of the ECL intensity of Ru(bpy)₃²⁺ due to the increasing distance between Fc and the electrode surface. The combination of LPS and AS suppressed the formation of dsDNA, inhibited the activation of CRISPR-Cas12a, and prevented further cleavage of ssDNA-Fc. This mechanism aided in upholding the integrity of ssDNA-Fc on the surface of the electrode and was combined with ECL quenching induced by the target. The ECL intensity decreased linearly as the concentration of LPS increased from 1 to 50,000 pg/mL and followed a logarithmic relationship. This method exhibited a remarkably low detection limit of 0.24 pg/mL, which meets the requirement for low-concentration detection of LPS in the human body. The proposed method demonstrates the capacity of CRISPR-Cas12a to perform non-specific cutting of single-stranded DNA and transform the resultant cutting substances into changes in the ECL signal. By amalgamating this approach with the distinct identification abilities of LPS and its aptamers, a simple, responsive, and discriminatory LPS assay was established that holds immense significance for clinical diagnosis.
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The outer membrane of gram-negative bacteria is primarily composed of lipopolysaccharide (LPS). In addition to protection, LPS defines the distinct serogroups used to identify bacteria specifically. Furthermore, LPS also act as highly potent stimulators of innate immune cells, a phenomenon essential to understanding pathogen invasion in the body. The complex multi-step process of LPS binding to cells involves several binding partners, including LPS binding protein (LBP), CD14 in both membrane-bound and soluble forms, membrane protein MD-2, and toll-like receptor 4 (TLR4). Once these pathways are activated, pro-inflammatory cytokines are eventually expressed. These binding events are also affected by the presence of monomeric or aggregated LPS.
Traditional techniques to detect LPS include the rabbit pyrogen test, the monocyte activation test and Limulus-based tests. Modern approaches are based on protein, antibodies or aptamer binding. Recently, novel techniques including electrochemical methods, HPLC, quartz crystal microbalance (QCM), and molecular imprinting have been developed. These approaches often use nanomaterials such as gold nanoparticles, quantum dots, nanotubes, and magnetic nanoparticles.
This chapter reviews current developments in endotoxin detection with a focus on modern novel techniques that use various sensing components, ranging from natural biomolecules to synthetic materials. Highly integrated and miniaturized commercial endotoxin detection devices offer a variety of options as the scientific and technologic revolution proceeds.
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To detect endotoxin rapidly and sensitively, a sandwich surface acoustic wave (SAW) sensing strategy based on the sensing interface Ti3C2Tx/Au NPs/Apt1/MCH and the nanoprobe Au NPs/Apt2/mPEG was proposed. In addition, a microfluidic chip integrated with functional parts of derivatization, capture, enrichment, and detection was designed and fabricated. The sensing interface was designed to improve the acoustic properties of the SAW sensor and capture the target. The nanoprobe was prepared and applied for specific endotoxin binding and phase signal amplification. It was illustrated that the sensitivity for the on-chip detection of endotoxin was improved and the detection limit was reduced by one order of magnitude by using the newly-constructed sensing strategy. Moreover, the endotoxin conjugate was concentrated in a continuous flow mode on the sensing interface of the SAW sensor, and the secondary amplification of the phase signal was achieved, which was approximately 1.5 times higher than that obtained in the static mode. A novel in situ quantitative detection strategy for endotoxin was thus established, and it was applied to measure endotoxin in real serum samples with a detection limit of 0.05 ng/mL and a linear range between 0.05 and 10 ng/mL.
Simple and fast colorimetric detection of lipopolysaccharide based on aptamer and SYBR Green I mediated aggregation of gold nanoparticles
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Citation Excerpt :
In recent years, great progress has been made in the development of biosensors for LPS detection, using a variety of LPS-specific probes including peptides [15], proteins [16], antibodies [17] and aptamers [18]. Among these, aptamer is regarded as an outstanding probe because of its low cost, high specificity and stability, ease of synthesis and very little difference between batches [19–22]. Since the LPS binding aptamer (LBA) was first selected by Kim et al. in 2012 [19], a great deal of LBA-based biosensors have been developed for LPS detection [20,22].
Lipopolysaccharide (LPS) poses a considerable threat to food safety and human health. A colorimetric assay for LPS detection based on LPS binding aptamer (LBA) and SYBR Green I (SG) mediated aggregation of gold nanoparticles (AuNPs) was established. In the absence of LPS, the LBA was absorbed onto the AuNPs surface which prevented SG-induced aggregation of AuNPs, and the sensing system exhibited red color. When LPS was added, it interacted with the LBA, forming a complex. At higher LPS concentration, many LBAs were exhausted resulting in SG-induced aggregation of AuNPs, and color change from red to blue. The range of colorimetric detection of LPS was linear in 0–12 EU/mL, with a limit of detection of 0.1698 EU/mL. Spiked LPS in real samples and interfering substances were also identified. This assay ingeniously using the fluorescent dye SG as an effective trigger of AuNPs aggregation, is rapid and facile than most of those earlier reported LBA-based LPS assays, and there is potential to be modified to construct assays for other targets.
A novel labeled and label-free dual electrochemical detection of endotoxin based on aptamer-conjugated magnetic reduced graphene oxide-gold nanocomposite
2022, Journal of Electroanalytical Chemistry
Lipopolysaccharide (LPS) or endotoxin is an amphiphilic macromolecule found in the outer membrane of gram-negative bacteria. Even low amounts of LPS could result in death. Therefore, sensitive detection of endotoxin in the range of ng.mL⁻¹ is vital. The dual electrochemical detection of endotoxin was designed. Thiourea was utilized to conjugate the magnetic graphene-based nanocomposite to a pair of amino-modified aptamers to increase the selectivity and sensitivity through the increase of active space occupied by aptamers attached to the nanocomposite. X-ray diffraction, FT-IR spectroscopy, Raman spectroscopy, VSM, FE-SEM, and TEM were applied to study nanomaterials. Electrochemical techniques, including cyclic voltammetry (CV), Square‐wave voltammetry (SWV) and Electrochemical Impedance Spectroscopy (EIS) were accomplished to study electrochemical properties of the aptasensor. Both labeled, and label-free electrochemical detection of endotoxin were performed using methylene blue and ferrocyanide, respectively. The aptasensor exhibits the limit of detection as low as 4 fg/mL and 0.2 fg/mL, with linear range of detection 0.1–0.9 pg.mL⁻¹ and 0.01–0.09 pg.mL⁻¹ for ferrocyanide and methylene blue, respectively. The aptasensor showed a considerable response to LPS over other biomolecules like glucose. This aptasensor exhibited a good response to the serum of patient and normal people, which certificates its practicality in medical aims.

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¹: These authors equally contributed to this work.

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Harnessing aptamers for electrochemical detection of endotoxin

Abstract

Section snippets

Materials

Selection of LPS affinity aptamers

Conclusions

Acknowledgments

Biochem. Biophys. Res. Commun.

J. Immunol. Methods

J. Chromatogr. A

J. Chromatogr. A

J. Immunol. Methods

J. Biotechnol.

J. Mol. Biol.

Biochem. Biophys. Res. Commun.

Sens. Actuators B

TrAC Trends Anal. Chem.

Biosens. Bioelectron.

Biosens. Bioelectron.

Biosens. Bioelectron.

J. Chromatogr. A

Colloids Surf. B

Synth. Met.

J. Colloid Interface Sci.

Biosens. Bioelectron.

The lipooligosaccharides of pathogenic gram-negative bacteria

Crit. Rev. Microbiol.

Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin

Annu. Rev. Immunol.