Sonoprinting nanoparticles on cellular spheroids via surface acoustic waves for enhanced nanotherapeutics delivery.

Nanotherapeutics, on their path to the target tissues, face numerous physicochemical hindrances that affect their therapeutic efficacy. Physical barriers become more pronounced in pathological tissues, such as solid tumors, where they limit the penetration of nanocarriers into deeper regions, thereby preventing the efficient delivery of drug cargo. To address this challenge, we introduce a novel approach that employs surface acoustic wave (SAW) technology to sonoprint and enhance the delivery of nanoparticles onto and into cell spheroids. Our SAW platform is designed to generate focused and unidirectional acoustic waves for creating vigorous acoustic streaming while promoting Bjerknes forces. The effect of SAW excitation on cell viability, as well as the accumulation and penetration of nanoparticles on human breast cancer (MCF 7) and mouse melanoma (YUMM 1.7) cell spheroids were investigated. The high frequency, low input voltage, and contact-free nature of the proposed SAW system ensured over 92% cell viability for both cell lines after SAW exposure. SAW sonoprinting enhanced the accumulation of 100 nm polystyrene particles on the periphery of the spheroids to near four-fold, while the penetration of nanoparticles into the core regions of the spheroids was improved up to three times. To demonstrate the effectiveness of our SAW platform on the efficacy of nanotherapeutics, the platform was used to deliver nanoliposomes encapsulated with the anti-cancer metal compound copper diethyldithiocarbamate (CuET) to MCF 7 and YUMM 1.7 cell spheroids. A three-fold increase in the cytotoxic activity of the drug was observed in spheroids under the effect of SAW, compared to controls. The capacity of SAW-based devices to be manufactured as minuscule wearable patches can offer highly controllable, localized, and continuous acoustic waves to enhance drug delivery efficiency to target tissues.

Acoustofluidics - changing paradigm in tissue engineering, therapeutics development, and biosensing.

For more than 70 years, acoustic waves have been used to screen, diagnose, and treat patients in hundreds of medical devices. The biocompatible nature of acoustic waves, their non-invasive and contactless operation, and their compatibility with wide visualization techniques are just a few of the many features that lead to the clinical success of sound-powered devices. The development of microelectromechanical systems and fabrication technologies in the past two decades reignited the spark of acoustics in the discovery of unique microscale bio applications. Acoustofluidics, the combination of acoustic waves and fluid mechanics in the nano and micro-realm, allowed researchers to access high-resolution and controllable manipulation and sensing tools for particle separation, isolation and enrichment, patterning of cells and bioparticles, fluid handling, and point of care biosensing strategies. This versatility and attractiveness of acoustofluidics have led to the rapid expansion of platforms and methods, making it also challenging for users to select the best acoustic technology. Depending on the setup, acoustic devices can offer a diverse level of biocompatibility, throughput, versatility, and sensitivity, where each of these considerations can become the design priority based on the application. In this paper, we aim to overview the recent advancements of acoustofluidics in the multifaceted fields of regenerative medicine, therapeutic development, and diagnosis and provide researchers with the necessary information needed to choose the best-suited acoustic technology for their application. Moreover, the effect of acoustofluidic systems on phenotypic behavior of living organisms are investigated. The review starts with a brief explanation of acoustofluidic principles, the different working mechanisms, and the advantages or challenges of commonly used platforms based on the state-of-the-art design features of acoustofluidic technologies. Finally, we present an outlook of potential trends, the areas to be explored, and the challenges that need to be overcome in developing acoustofluidic platforms that can echo the clinical success of conventional ultrasound-based devices.

Development and mechanical characterization of decellularized scaffolds for an active aortic graft.

Decellularized porcine aortas are proposed as scaffolds for revolutionary active aortic grafts. A change in the static and dynamic mechanical properties, associated with the microstructure of elastin and collagen fibers, corresponds to alteration in the cyclic expansion and perfusion, in addition to possible graft damage. Therefore, the present study thoroughly investigates the mechanical response of the decellularized scaffolds of human and porcine origin to static and dynamic mechanical loads. The responses of the native human and porcine aortas are also compared; this is unavailable in the literature. Because the aorta is subjected to pulsatile blood pressure, dynamical responses to cyclic loads and their associated viscoelastic properties are particularly relevant for advanced graft design. In parallel, this study examines the microstructure of the decellularized aorta. The resulting data are compared to the analogous data obtained for the native human and porcine tissues. The results indicate that by using an optimized decellularization protocol - based on sodium dodecyl sulfate (SDS) and DNase - that minimizes mechanical and structural changes of the tissue, layered scaffolds with static and dynamic properties very similar to natural human aortas are obtained. In particular, a decellularized porcine aorta is non-inferior to a decellularized human aorta. STATEMENT OF SIGNIFICANCE: About 55,000 patients undergo abdominal aortic aneurysm repair annually in the USA. The currently implanted grafts present a large mechanical mismatch with the native tissue. This increases the pulsatile nature of the blood flow with negative consequences to the organ perfusion. For this reason, biomimetic and mechanically compatible grafts for aortic repair are urgently needed and they can be obtained through tissue engineering. In this study, scaffolds from porcine and human aortas are obtained from an optimized decellularization protocol. They are accurately compared to the native tissue and present the ideal static and dynamic mechanical properties for developing innovative aortic grafts.

A 6-bromoindirubin-3'-oxime incorporated chitosan-based hydrogel scaffold for potential osteogenic differentiation: Investigation of material properties in vitro.

Effective treatments for critical size bone defects remain challenging. 6-Bromoindirubin-3'-Oxime (BIO), a glycogen synthase kinase 3ß inhibitor, is a promising alternative for treatment of these defects since it aids in promoting osteogenic differentiation. In this study, BIO is incorporated into a new formulation of the guanosine diphosphate cross-linked chitosan scaffold to promote osteogenic differentiation. BIO incorporation was confirmed with 13C NMR through a novel concentration dependent peak around 41 ppm. The rapid gelation rate was maintained along with the internal structure's stability. The 10 µM BIO dose supported the control scaffold's microstructure demonstrating a suitable porosity and a low closed pore percentage. While pore sizes of BIO incorporated scaffolds were slightly smaller, pore heterogeneity was maintained. A proof-of-concept study with C2C12 cells suggested a dose-dependent response of BIO on early stages of osteogenic differentiation within the scaffold. These results support future work to examine BIO's role on osteogenic differentiation and biomineralization of encapsulated cells in the scaffold for bone regeneration.

INGAP-Peptide Variants as a Novel Therapy for Type 1 Diabetes: Effect on Human Islet Insulin Secretion and Gene Expression.

Islet transplantation offers a long-term cure for Type 1 Diabetes (T1D), freeing patients from daily insulin injections. Therapeutic peptides have shown potential to increase the insulin output of pancreatic islets, maximizing the impact of grafted cells. The islet neogenesis-associated protein (INGAP), and its bioactive core (INGAP-P), stimulate beta-cell function and viability, offering the possibility for islet treatment prior to implant. However, dosing efficacy is limited by low circulation time and enzyme degradation. This proof-of-concept study presents the investigation of novel molecular variants of INGAP-P to find a more bioactive form. Custom-designed peptide variants of INGAP-P were synthesized and tested for their effect on the insulin secretion and gene expression of live human islets. We exposed the live islets of five donors to varying glucose concentrations with INGAP-P variants in solution. We identified four peptide variants (I9, I15Tyr, I19 and I15Cys) which displayed statistically significant enhancements over negative controls (representing a 1.6-2.8-fold increase in stimulation index). This is the first study that has assessed these INGAP-P variants in human islets. It highlights the potential for customized peptides for type 1 diabetes therapy and provides a foundation for future peptide-screening experiments.

Decellularized extracellular matrix: New promising and challenging biomaterials for regenerative medicine.

Extracellular matrix is rich in biomolecules including structural proteins, glycosaminoglycans, and small molecules that are important for the maintenance and repair of tissue. Decellularized extracellular matrix (dECM) is expected to retain these key biomolecules and makes it a promising biomaterial candidate for regenerative medicine applications. To date, dECM-particle based biomaterials have been developed to engineer over 15 tissue types or organs, with the ultimate goal of mimicking specific biological and physical properties of the native tissue. The most common scaffold types are injectable hydrogels, electrospun scaffolds and bioprinted scaffolds. The purpose of this review paper is to highlight key challenges, fabrication methods and progress made for each tissue type, along with the discussion of other elements that are integral to push dECM biomaterials towards effective and specialized tissue repair.

In vitro models for head and neck cancer: Current status and future perspective.

The 5-year overall survival rate remains approximately 50% for head and neck (H&N) cancer patients, even though new cancer drugs have been approved for clinical use since 2016. Cancer drug studies are now moving toward the use of three-dimensional culture models for better emulating the unique tumor microenvironment (TME) and better predicting in vivo response to cancer treatments. Distinctive TME features, such as tumor geometry, heterogenous cellularity, and hypoxic cues, notably affect tissue aggressiveness and drug resistance. However, these features have not been fully incorporated into in vitro H&N cancer models. This review paper aims to provide a scholarly assessment of the designs, contributions, and limitations of in vitro models in H&N cancer drug research. We first review the TME features of H&N cancer that are most relevant to in vitro drug evaluation. We then evaluate a selection of advanced culture models, namely, spheroids, organotypic models, and microfluidic chips, in their applications for H&N cancer drug research. Lastly, we propose future opportunities of in vitro H&N cancer research in the prospects of high-throughput drug screening and patient-specific drug evaluation.

Enhancing metabolic activity and differentiation potential in adipose mesenchymal stem cells via high-resolution surface-acoustic-wave contactless patterning.

Acoustofluidics has shown great potential for label-free bioparticle patterning with excellent biocompatibility. Acoustofluidic patterning enables the induction of cell-cell interactions, which play fundamental roles in organogenesis and tissue development. One of the current challenges in tissue engineering is not only the control of the spatial arrangement of cells but also the preservation of cell patterns over time. In this work, we developed a standing surface acoustic wave-based platform and demonstrated its capability for the well-controlled and rapid cell patterning of adipose-derived mesenchymal stem cells in a high-density homogenous collagen hydrogel. This biocompatible hydrogel is easily UV crosslinked and can be retrieved within 3 min. Acoustic waves successfully guided the cells toward pressure nodal lines, creating a contactless alignment of cells in <5 s in culture media and <1 min in the hydrogel. The acoustically patterned cells in the hydrogel did not show a decrease in cell viability (>90%) 48 h after acoustic induction. Moreover, 45.53% and 30.85% increases in metabolic activity were observed in growth and differentiation media, respectively, on Day 7. On Day 14, a 32.03% change in metabolic activity was observed using growth media, and no significant difference was observed using differentiation media. The alkaline phosphatase activity showed an increase of 80.89% and 24.90% on Days 7 and 14, respectively, for the acoustically patterned cells in the hydrogel. These results confirm the preservation of cellular viability and improved cellular functionality using the proposed high-resolution acoustic patterning technique and introduce unique opportunities for the application of stem cell regenerative patches for the emerging field of tissue engineering.

One-Step Synthesis of Nanoliposomal Copper Diethyldithiocarbamate and Its Assessment for Cancer Therapy.

The metal complex copper diethyldithiocarbamate (CuET) induces cancer cell death by inhibiting protein degradation and induces proteotoxic stress, making CuET a promising cancer therapeutic. However, no clinical formulation of CuET exists to date as the drug is insoluble in water and exhibits poor bioavailability. To develop a scalable formulation, nanoliposomal (LP) CuET was synthesized using ethanol injection as a facile one-step method that is suitable for large-scale manufacturing. The nanoparticles are monodispersed, colloidally stable, and approximately 100 nm in diameter with an encapsulation efficiency of over 80%. LP-CuET demonstrates excellent stability in plasma, minimal size change, and little drug release after six-month storage at various temperatures. Additionally, melanoma cell lines exhibit significant sensitivity to LP-CuET and cellular uptake occurs predominantly through endocytosis in YUMM 1.7 cancer cells. Intracellular drug delivery is mediated by vesicle acidification with more nanoparticles being internalized by melanoma cells compared with RAW 264.7 macrophages. Additionally, the nanoparticles preferentially accumulate in YUMM 1.7 tumors where they induce cancer cell death in vivo. The development and characterization of a stable and scalable CuET formulation illustrated in this study fulfils the requirements needed for a potent clinical grade formulation.

Design and development of Branched Poly(ß-aminoester) nanoparticles for Interleukin-10 gene delivery in a mouse model of atherosclerosis.

Atherosclerosis progression is a result of chronic and non-resolving inflammation, effective treatments for which still remain to be developed. We designed and developed branched poly(ß-amino ester) nanoparticles (NPs) containing plasmid DNA encoding IL-10, a potent anti-inflammatory cytokine to atherosclerosis. The NPs (NP-VHPK) are functionalized with a targeting peptide (VHPK) specific for VCAM-1, which is overexpressed by endothelial cells at sites of atherosclerotic plaque. The anionic coating affords NP-VHPK with significantly lower toxicity than uncoated NPs in both endothelial cells and red blood cells (RBCs). Following injection of NP-VHPK in ApoE-/- mice, Cy5-labelled IL-10 significantly accumulates in both whole aortas and aortic sinus sections containing plaque compared to injection with a non-targeted control. Furthermore, IL-10 gene delivery results in an attenuation of inflammation locally at the plaque site. NP-VHPK may thus have the potential to reduce the inflammatory component of atherosclerosis in a safe and effective manner. STATEMENT OF SIGNIFICANCE: Atherosclerosis is a chronic inflammatory disease that results in the formation of lipid-laden plaques within vascular walls. Although treatments using drugs and antibodies are now beginning to address the inflammation in atherosclerosis, neither is sufficient for long-term therapy. In this paper, we introduce a strategy to deliver genes encoding the anti-inflammatory protein interleukin-10 (IL-10) in vivo. We showed that Branched Poly(ß-aminoester) carrying the IL-10 gene are able to localize specifically at the plaque via surface-functionalized targeting moieties against inflamed VCAM-1 and/or ICAM-1 and to facilitate gene transcription by ECs to increase the local concentration of the IL-10 within the plaque. To date, there is no report involving non-viral nanotechnology to provide gene-based therapies for atherosclerosis.

Encapsulation and differentiation of adipose-derived mesenchymal stem cells in a biomimetic purine cross-linked chitosan sponge.

Mesenchymal stem cells derived from adipose tissue have become a widely investigated cell source to use in tissue engineering applications. However, an optimal delivery scaffold for these cells is still needed. A rapidly gelling, injectable chitosan sponge was proposed in this study as a potential candidate for a suitable delivery scaffold. The results demonstrated the ability to encapsulate the stem cells at a 97.6% encapsulation efficiency and that the cells maintain their viability within the sponge. With the potential of using this scaffold for bone tissue engineering, ALP activity assay and fluorescent imaging for osteocalcin proved the ability to differentiate the encapsulated cells into the osteogenic lineage. Furthermore, co-encapsulation of pyrophosphatase within the sponge was investigated as a method to overcome the inhibitory effects that the sponge degradation by-products have on mineralization. Alizarin Red S staining demonstrated the beneficial effects of adding pyrophosphatase, where a significant increase in mineralization levels was achieved.

3D Printed In Vitro Dentin Model to Investigate Occlusive Agents against Tooth Sensitivity.

Tooth sensitivity is a painful and very common problem. Often stimulated by consuming hot, cold, sweet, or acidic foods, it is associated with exposed dentin microtubules that are open to dental pulp. One common treatment for tooth hypersensitivity is the application of occlusive particles to block dentin microtubules. The primary methodology currently used to test the penetration and occlusion of particles into dentin pores relies upon dentin discs cut from extracted bovine/human teeth. However, this method is limited due to low accessibility to the raw material. Thus, there is a need for an in vitro dentin model to characterize the effectiveness of occlusive agents. Three-dimensional printing technologies have emerged that make the printing of dentin-like structures possible. This study sought to develop and print a biomaterial ink that mimicked the natural composition and structure of dentin tubules. A formulation of type I collagen (Col), nanocrystalline hydroxyapatite (HAp), and alginate (Alg) was found to be suitable for the 3D printing of scaffolds. The performance of the 3D printed dentin model was compared to the natural dentin disk by image analysis via scanning electron microscopy (SEM), both pre- and post-treatment with occlusive microparticles, to evaluate the degree of dentinal tubule occlusion. The cytocompatibility of printed scaffolds was also confirmed in vitro. This is a promising biomaterial system for the 3D printing of dentin mimics.

Rapid Formation of Multicellular Spheroids in Boundary-Driven Acoustic Microstreams.

3D cell spheroid culture has emerged as a more faithful recreation of cell growth environment compared to conventional 2D culture, as it can maintain tissue structures, physicochemical characteristics, and cell phenotypes. The majority of current spheroid formation methods are limited to a physical agglomeration of the desired cell type, and then relying on cell capacity to secrete extracellular matrix to form coherent spheroids. Hence, apart from being time-consuming, their success in leading to functional spheroid formation is also cell-type dependent. In this study, a boundary-driven acoustic microstreaming tool is presented that can simultaneously congregate cells and generate sturdy cell clusters through incorporating a bioadhesive such as collagen for rapid production of spheroids. The optimized mixture of type I collagen (0.42 mg mL-1 ) and methylcellulose (0.4% w/v ) accelerates the coagulation of cell-matrix as fast as 10 s while avoiding their adhesion to the device, and thereby offering easy spheroid retrieval. The versatility of the platform is shown for the production of MDA-MB-231 and MCF-7 spheroids, multicellular spheroids, and composite spheroids made of cells and microparticles. The ability to produce densely packed spheroids embedded within a biomimetic extracellular matrix component, along with rapid formation and easy collection of spheroids render the proposed device a step in technology development required to realize potentials of 3D constructs such as building blocks for the emerging field of bottom-up tissue engineering.

Viscous Core Liposomes Increase siRNA Encapsulation and Provides Gene Inhibition When Slightly Positively Charged.

Since its discovery, evidence that siRNA was able to act as an RNA interference effector, led to its acceptation as a novel medicine. The siRNA approach is very effective, due to its catalytic mechanism, but still the limitations of its cellular delivery should be addressed. One promising form of non-viral gene delivery system is liposomes. The variable and versatile nature of the lipids keeps the possibility to upgrade the liposomal structure, which makes them suitable for encapsulation and delivery of drugs. However, to avoid the limitation of fast release for the hydrophilic drug, we previously designed viscous core liposomes. We aimed in this work to evaluate if these viscous core liposomes (NvcLs) could be of interest for siRNA encapsulation. Then, we sought to add a limited amount of positive charges to provide cell interaction and transfection. Cationic lipid dimyristoylaminopropylaminopropyl or the polymer poly(ethylenimine) were incorporated in NvcL to produce positively charged viscous core liposomes (PvcL) by a customized microfluidic device. We found that NvcLs increased the encapsulation efficiency and loading content with regards to the neutral liposome. Both PvcLPEI and PvcLDMAPAP exhibited transfection and GFP knock-down (≈40%) in both 2D and 3D cell cultures. Finally, the addition of slight positive charges did not induce cell toxicity.

Mussel-inspired antimicrobial coating on PTFE barrier membranes for guided tissue regeneration.

Guided tissue regeneration procedures to treat periodontitis lesions making use of polytetrafluoroethylene (PTFE) membranes exhibit large variability in their surgical outcomes, due to bacterial infection following implantation. This work reports on a facile method to obtain antimicrobial coatings for such PTFE membranes, by exploiting a mussel-inspired approach andin-situformation of silver nanoparticles (AgNPs). PTFE films were initially coated with self-polymerized 3,4-dihydroxy-DL-phenylalanine (DOPA) (PTFE-DOPA), then incubated with AgNO3solution. In the presence of catechol moieties, Ag+ions reduced into Ag0, forming AgNPs of around 68 nm in the polyDOPA coating on PTFE membranes (PTFE-DOPA-Ag). The x-ray photoelectron spectroscopy, atomic force microscopy and scanning electron microscopy analyses indicated that the AgNPs were distributed quite homogeneously in the polymeric membrane. The antimicrobial ability of PTFE-DOPA-Ag membranes againstStaphylococcus aureusandEscherichia coliwas assessed.In vitrocell assay using NIH 3T3 fibroblasts showed that, although cells were adhered to PTFE-DOPA-Ag membranes, their viability and proliferation were limited demonstrating again the antibacterial activities of PTFE-DOPA-Ag membranes. This work provides proof-of-concept study of a new versatile approach for AgNPs coating, which may be easily applied to many other types of polymeric or metallic implants through exploiting the adhesive behavior of mussel-inspired coatings.

A core-shell guanosine diphosphate crosslinked chitosan scaffold as a potential co-encapsulation platform.

Recent engineering strategies to better mimic native tissue architecture involve co-encapsulation of cell lineages and/or growth factors in multi-compartmental scaffolds. This study introduces a core-shell platform based on a rapidly gelling guanosine diphosphate cross-linked chitosan scaffold for co-culture. The core-shell sponge is fabricated through combination of chitosan and guanosine diphosphate in 3 steps with each shell layer deposited around the previous layer. Co-encapsulation of pre-osteoblastic MC-3T3 cells and growth factors in the core-shell sponge showed similar microstructure to the standard sponge with high pore connectivity and low closed porosity (<0.4 %). A viable cell population was maintained over time with enhanced cellular functionality when ascorbic acid was added in the same compartment. Co-culture was explored with a proof-of-concept study shown for MC-3T3 and endothelial cells showing homogeneous distribution of cells in their intended compartment. Overall, this core-shell scaffold shows potential as a platform for the regeneration of multiple tissues.

Synthesis and Screening of Novel Peptides on Human Pancreatic Islets for Type 1 Diabetes Therapies.

Type 1 diabetic patients characteristically exhibit a loss of insulin production, leading to chronic hyperglycemia and related complications. Herein we describe the design, synthesis and screening of novel oligopeptides for their potential to enhance the secretion of insulin from human pancreatic islets. The investigation of these compounds, based off the patented INGAP-PP sequence, aims to identify the peptide features key to maximizing insulin secretion.Clinical Relevance - This report describes the relative efficacy of selected novel compounds for potential Type 1 Diabetes Therapy. Tested on live human pancreatic islets, the compounds are evaluated for their enhancing/inhibitory effect on the secretion of insulin. These studies pave the way for future targeted drug therapies.

Low-Cost Graphene-Based Digital Microfluidic System.

In this work, the laser-scribing technique was used as a low-cost, rapid and facile method for fabricating digital microfluidic (DMF) systems. Laser-scribed graphene (LSG) electrodes are directly synthesized on flexible substrates to pattern the DMF electrode arrays. This facilitates the DMF electrodes' fabrication process by eliminating many microfabrication steps. An electrowetting test was performed to investigate the effectiveness of the LSG DMF electrodes in changing the contact angles of droplets. Different DMF operations were successfully performed using the proposed LSG DMF chips in both open and closed DMF systems. The quality and output resolution were examined to assess the performance of such patterned electrodes in the DMF systems. To verify the efficacy of the LSG DMF chips, a one-step direct assay for the detection of Legionellapneumophila deoxyribonucleic acid (DNA) was performed on the chip without the need for any washing step. The high specificity in distinguishing a single-nucleotide mismatch was achieved by detecting target DNA concentrations as low as 1 nM. Our findings suggest that the proposed rapid and easy fabrication method for LSG DMF electrodes offers a great platform for low-cost and easily accessible point-of-care diagnostic devices.

In vitro and in vivo investigation of osteogenic properties of self-contained phosphate-releasing injectable purine-crosslinked chitosan-hydroxyapatite constructs.

Bone fracture repair is a multifaceted, coordinated physiological process that requires new bone formation and resorption, eventually returning the fractured bone to its original state. Currently, a variety of different approaches are pursued to accelerate the repair of defective bones, which include the use of 'gold standard' autologous bone grafts. However, such grafts may not be readily available, and procedural complications may result in undesired outcomes. Considering the ease of use and tremendous customization potentials, synthetic materials may become a more suitable alternative of bone grafts. In this study, we examined the osteogenic potential of guanosine 5'-diphosphate-crosslinked chitosan scaffolds with the incorporation of hydroxyapatite, with or without pyrophosphatase activity, both in vitro and in vivo. First, scaffolds embedded with cells were characterized for cell morphology, viability, and attachment. The cell-laden scaffolds were found to significantly enhance proliferation for up to threefold, double alkaline phosphatase activity and osterix expression, and increase calcium phosphate deposits in vitro. Next, chitosan scaffolds were implanted at the fracture site in a mouse model of intramedullary rod-fixed tibial fracture. Our results showed increased callus formation at the fracture site with the scaffold carrying both hydroxyapatite and pyrophosphatase in comparison to the control scaffolds lacking both pyrophosphatase and hydroxyapatite, or pyrophosphatase alone. These results indicate that the pyrophosphatase-hydroxyapatite composite scaffold has a promising capacity to facilitate bone fracture healing.

Identification of two aptamers binding to Legionella pneumophila with high affinity and specificity.

Legionella pneumophila (Lp) is a water borne bacterium causing Legionnaires' Disease (LD) in humans. Rapid detection of Lp in water system is essential to reduce the risk of LD outbreaks. The methods currently available require expert skills and are time intensive, thus delaying intervention. In situ detection of Lp by biosensor would allow rapid implementation of control strategies. To this end, a biorecognition element is required. Aptamers are considered promising biorecognition molecules for biosensing. Aptamers are short oligonucleotide sequence folding into a specific structure and are able to bind to specific molecules. Currently, no aptamer and thus no aptamer-based technology exists for the detection of Lp. In this study, Systemic Evolution of Ligands through EXponential enrichment (SELEX) was used to identify aptamers binding specifically to Lp. Ten rounds of positive selection and two rounds of counter-selection against two Pseudomonas species were performed. Two aptamers binding strongly to Lp were identified with KD of 116 and 135 nM. Binding specificity of these two aptamers to Lp was confirmed by flow cytometry and fluorescence microscopy. Therefore, these two aptamers are promising biorecognition molecules for the detection of Lp in water systems.