Soil invertebrate toxicity and bioaccumulation of nano copper oxide and copper sulphate in soils, with and without biosolids amendment.

The fate, toxicity and bioaccumulation of copper oxide nanoparticles (nCuO) was investigated in soil, with and without biosolids amendment, through chronic exposures using the earthworm, Eisenia andrei, and the collembolan, Folsomia candida. The effects of copper sulphate (CuSO4) were included so as to compare the behavior of nCuO to a readily soluble counterpart. The fate of nCuO was evaluated through characterization of dissolved and nano-particulate fractions (via single particle ICP-MS) as well as extractable Cu2+ throughout the duration of select tests. Neither Cu form was particularly toxic to F. candida, but effects on E. andrei reproduction were significant in all treatments (IC50 range: 98 - 149 mg Cu kg-1 dry soil). There were no significant differences in toxicity between the Cu forms, nor in extractable Cu2+ activities, indicative that particle dissolution within the soil and, subsequent activity of Cu2+ was likely the primary mode of toxicity in the nCuO exposures. The presence of biosolids did not significantly alter toxicity of nCuO, but did affect Cu2+ activity over time. Bioaccumulation of total Cu in E. andrei when exposed to nCuO (kinetic bioaccumulation factor (BAFk): 0.80 with biosolids and 0.81 without) was lower than exposure to CuSO4 (BAFk: 2.31 with biosolids and 1.12 without). Enhanced dark-field hyperspectral imaging showed accumulation of nCuO along the epidermis and gut of E. andrei, with trace amounts observed in muscle and chloragogenous tissue, providing evidence of nCuO translocation within the organism. The present study demonstrates that the current risk assessment approach for trace metals in the environment, based on substance solubility and bioavailability of the dissolved free ion, are applicable for nCuO exposure to soil invertebrates, but that the rate of particle dissolution in different soil environments is an important factor for consideration.

Development and Evaluation of a Quantitative Fluorescent Lateral Flow Immunoassay for Cystatin-C, a Renal Dysfunction Biomarker.

The diagnosis, prognosis, and control of chronic kidney disease rely on an understanding of the glomerular filtration rate (GFR). The renal clearance of the cystatin-C is closely associated with the GFR. Cystatin-C is a more suitable GFR marker than the commonly used creatinine. General techniques for cystatin-C calculation, such as particle-enhanced turbidimetric and nephelometric assay, are time-consuming and tedious. Here, we propose a rapid, quantitative immunoassay for the detection of cystatin-C. A fluorescence-based lateral-flow kit was developed in a sandwich format by using a monoclonal antibody. A Linear calibration was obtained over the clinical diagnostic range of 0.023-32 µg/mL and the limit of detection (LOD) was 0.023 µg/mL and the limit of quantification (LOQ) was 0.029 µg/mL. Average recoveries from spiked urine samples ranged from 96-100% and the coefficient of variation was less than 4% for both intra and inter-day assays with excellent repeatability. With the comparison with an ELISA kit, the developed kit is highly sensitive, performs well over the detection range, provides repeatable results in a short time, and can easily be used at point-of-care (POC), making it an ideal candidate for rapid testing in early detection, community screening for renal function disorders.

A review of Cryptosporidium spp. and their detection in water.

Cryptosporidium spp. are one of the most important waterborne pathogens worldwide and a leading cause of mortality from waterborne gastrointestinal diseases. Detection of Cryptosporidium spp. in water can be very challenging due to their low numbers and the complexity of the water matrix. This review describes the biology of Cryptosporidium spp. and current methods used in their detection with a focus on C. parvum and C. hominis. Among the methods discussed and compared are microscopy, immunology-based methods using monoclonal antibodies, molecular methods including PCR (polymerase chain reaction)-based assays, and emerging aptamer-based methods. These methods have different capabilities and limitations, but one common challenge is the need for better sensitivity and specificity, particularly in the presence of contaminants. The application of DNA aptamers in the detection of Cryptosporidium spp. oocysts shows promise in overcoming these challenges, and there will likely be significant developments in aptamer-based sensors in the near future.

Assessment of Aptamer-Targeted Contrast Agents for Monitoring of Blood Clots in Computed Tomography and Fluoroscopy Imaging.

Objective: Random formation of thrombi is classified as a pathological process that may result in partial or complete obstruction of blood flow and limited perfusion. Further complications include pulmonary embolism, thrombosis-induced myocardial infraction, ischemic stroke, and others. Location and full delineation of the blood clot are considered to be two clinically relevant aspects that could streamline proper diagnosis and treatment follow-up. In this work, we prepared two types of X-ray attenuating contrast formulations, using fibrinogen aptamer as the clot-seeking moiety. Methods: Two novel aptamer-targeted formulations were designed. Iodine-modified bases were directly incorporated into a fibrinogen aptamer (iodo-FA). Isothermal titration calorimetry was used to confirm that these modifications did not negatively impact target binding. Iodo-FA was tested for its ability to produce concentration-dependent contrast enhancement in a phantom CT. It was subsequently tested in vitro with clotted human and swine blood. This allowed for translation into ex vivo testing, using fluoroscopy. FA was also used to functionalize gold nanoparticles (FA-AuNPs), and contrast capabilities were confirmed. This formulation was tested in vitro using clotted human blood in a CT scan. Results: Unmodified FA and iodo-FA demonstrated a nearly identical affinity toward fibrin, confirming that base modifications did not impact target binding. Iodo-FA and FA-AuNPs both demonstrated excellent concentration-dependent contrast enhancement capabilities (40.5 HU mM-1 and 563.6 HU µM-1, respectively), which were superior to the clinically available agent, iopamidol. In vitro CT testing revealed that iodo-FA is able to penetrate into the blood clots, producing contrast enhancement throughout, while FA-AuNPs only accumulated on the surface of the clot. Iodo-FA was thereby translated to ex vivo testing, confirming target-binding associated accumulation of the contrast material at the location of the clot within the dilation of the external carotid artery. This resulted in a 34% enhancement of the clot. Conclusions: Both iodo-FA and FA-AuNPs were confirmed to be effective contrast formulations in CT. Targeting of fibrin, a major structural constituent of thrombi, with these novel contrast agents would allow for higher contrast enhancement and better clot delineation in CT and fluoroscopy.

Multimodal plasmonic optical fiber grating aptasensor.

Tilted fiber Bragg gratings (TFBGs) are now a well-established technology in the scientific literature, bringing numerous advantages, especially for biodetection. Significant sensitivity improvements are achieved by exciting plasmon waves on their metal-coated surface. Nowadays, a large part of advances in this topic relies on new strategies aimed at providing sensitivity enhancements. In this work, TFBGs are produced in both single-mode and multimode telecommunication-grade optical fibers, and their relative performances are evaluated for refractometry and biosensing purposes. TFBGs are biofunctionalized with aptamers oriented against HER2 (Human Epidermal Growth Factor Receptor-2), a relevant protein biomarker for breast cancer diagnosis. In vitro assays confirm that the sensing performances of TFBGs in multimode fiber are higher or identical to those of their counterparts in single-mode fiber, respectively, when bulk refractometry or surface biosensing is considered. These observations are confirmed by numerical simulations. TFBGs in multimode fiber bring valuable practical assets, featuring a reduced spectral bandwidth for improved multiplexing possibilities enabling the detection of several biomarkers.

An aptamer-based colorimetric lateral flow assay for the detection of human epidermal growth factor receptor 2 (HER2).

An aptamer-based colorimetric lateral flow assay was developed for the detection of human epidermal growth factor receptor 2 (HER2). In this study, two approaches were examined using HER2 binding aptamers and gold nanoparticles. The first method used was a solution-based adsorption-desorption colorimetric approach wherein aptamers were adsorbed onto the gold nanoparticle surface. Upon the addition of HER2, HER2 binds specifically with its aptamer, releasing the gold nanoparticles. Addition of NaCl then induces the formation of gold nanoparticle aggregates. This leads to a color change from red to blue and a detection limit of 10 nM was achieved. The second method used an adsorption-desorption colorimetric lateral flow assay approach wherein biotin-modified aptamers were adsorbed onto the gold nanoparticle surface in the absence of HER2. In the presence of HER2, HER2 specifically binds with its aptamer leading to release of the gold nanoparticles. These solutions were applied to the lateral flow assay format and a detection limit of 20 nM was achieved. Both colorimetric and lateral flow assays are inexpensive, simple, rapid to perform and produce results visible to the naked-eye.

Correction to: Comparison of turn-on and ratiometric fluorescent G-quadruplex aptasensor approaches for the detection of ATP.

Regrettably, before online publication the figure of Scheme 2 has been pasted twice as Scheme 1.

Comparison of turn-on and ratiometric fluorescent G-quadruplex aptasensor approaches for the detection of ATP.

Two fluorescent aptasensor methods were developed for the detection of ATP in biochemical systems. The first method consisted of a label-free fluorescent "turn-on" approach using a guanine-rich ATP aptamer sequence and the DNA-binding agent berberine complex. In the presence of ATP, the ATP preferentially binds with its aptamer and conformationally changes into a G-quadruplex structure. The association of berberine with the G-quadruplex results in the enhancement of the fluorescence signal of the former. The detection limit of ATP was found to be 3.5 µM. Fluorescence, circular dichroism and melting temperature (Tm) experiments were carried out to confirm the binding specificity and structural changes. The second method employs the ratiometric fluorescent approach based on the Forster resonance energy transfer (FRET) for the detection of ATP using berberine along with a quencher (AuNRs, AgNPs) and a fluorophore (red quantum dots (RQDs), carbon dots (CDs)) labeled at 5' and 3' termini of the ATP-binding aptamer sequence. Upon addition of ATP and berberine, ATP specifically binds with its aptamer leading to the formation of G-quadruplex, and similarly, berberine also binds to the G-quadruplex. This leads to an enhancement of fluorescence of berberine while that of RQD and CDs were significantly quenched via FRET. The respective detection limits calculated were 3.6 µM and 3.8 µM, indicating these fluorescent aptasensor methods may be used for a wide variety of small molecules. Graphical abstract.

Label-free aptasensors based on fluorescent screening assays for the detection of Salmonella typhimurium.

We report two label-free fluorescent aptasensor methods for the detection of S. typhimurium. In the first method, we have used a ''turn off'' approach in which the aptamer is first intercalated with SYBR Green I (SG), leading to a greatly enhanced fluorescence signal. The addition of S. typhimurium (approximately 1530-96938 CFU/mL), which specifically binds with its aptamer and releases SG, leads to a linear decrease in fluorescence intensity. The lowest detection limit achieved with this approach was in the range of 733 CFU/mL. In the second method, a ''turn on'' approach was designed for S. typhimurium through the Förster resonance energy transfer (FRET) between Rhodamine B (RB) and gold nanoparticles (AuNPs). When the aptamer and AuNPs were mixed with RB, the fluorescence of RB was significantly quenched via FRET. The aptamer adsorbs to the AuNP surface to protect them from salt-induced aggregation, which leads to the fluorescence quenching of RB in presence of AuNPs. Upon the addition of S. typhimurium, S. typhimurium specifically binds with its aptamer and loses the capability to stabilize AuNPs. Thus, the salt easily induces the aggregation of AuNPs, resulting in the fluorescence recovery of the quenched RB. S. typhimurium concentrations ranging from 1530 to 96938 CFU/mL with the detection limit of 464 CFU/mL was achieved with this methodology. Given these data, some insights into the molecular interactions between the aptamer and the bacterial target are provided. These aptasensor methods also may be adapted for the detection of a wide variety of targets.

Lateral flow assays for Ochratoxin A using metal nanoparticles: comparison of "adsorption-desorption" approach to linkage inversion assembled nano-aptasensors (LIANA).

Nano-aptamer probes were prepared and used in lateral flow colorimetric assays for the detection of Ochratoxin A (OTA). In this study, two approaches were examined using 5'-biotin-modified OTA aptamers and silver or gold nanoparticles (AgNP or AuNP). The first method used an "adsorption-desorption" approach wherein aptamers were adsorbed onto the metal nanoparticle surface. Upon the addition of OTA, the aptamer binds specifically to the target, releasing the NPs. The above solutions were applied on a lateral flow assay (LFA) and a detection limit of 6.3 nM was achieved with both metal nanoparticles. The second method used a labelled approach based on Linkage Inversion Assembled Nano-Aptasensors (LIANAs) using a DNA linker containing a 5'-5' linkage inversion (5'-5' linker) to assemble biotinylated aptamer-functionalized metal nanoparticles. In the presence of target, OTA specifically binds with its aptamer leading to release of the linker and disassembly of LIANA aggregates into dispersed nanoparticles. The same solutions were applied in LFA format and the lowest detection limit of 0.63 nM was achieved. The results indicated that the LIANA-based LFA strips were more sensitive than the "adsoprtion-desorption" LFAs. Both lateral flow assays are inexpensive, simple, and rapid to perform and produces results visible to the naked-eye.

Preparation and characterization of aptamer-polyelectrolyte films and microcapsules for biosensing and delivery applications.

"Smart" materials are polymer systems that are able to change their physical or chemical properties in response to external stimuli in their environment. By adding a specific molecular recognition probe to a polymer, hybrid materials can be developed that retain the properties of the advanced polymer and gain the ability to respond to a specific molecular target. Aptamers are single-stranded oligonucleotides that are well-suited to serve as molecular recognition probes due to the specificity and affinity of their target recognition as well as their stability and ease of synthesis and labeling. In particular, their negatively charged backbone makes for their facile incorporation into polyelectrolyte-based materials. This article will provide a brief review of the currently reported biosensor and delivery platforms that have been reported employing aptamer-polyelectrolyte materials, as well as a detailed description of the methods used to synthesize and study films and microcapsules containing small-molecule aptamer probes.

Analysis of In Vitro Aptamer Selection Parameters.

Nucleic acid aptamers are novel molecular recognition tools that offer many advantages compared to their antibody and peptide-based counterparts. However, challenges associated with in vitro selection, characterization, and validation have limited their wide-spread use in the fields of diagnostics and therapeutics. Here, we extracted detailed information about aptamer selection experiments housed in the Aptamer Base, spanning over two decades, to perform the first parameter analysis of conditions used to identify and isolate aptamers de novo. We used information from 492 published SELEX experiments and studied the relationships between the nucleic acid library, target choice, selection methods, experimental conditions, and the affinity of the resulting aptamer candidates. Our findings highlight that the choice of target and selection template made the largest and most significant impact on the success of a de novo aptamer selection. Our results further emphasize the need for improved documentation and more thorough experimentation of SELEX criteria to determine their correlation with SELEX success.

Comprehensive analytical comparison of strategies used for small molecule aptamer evaluation.

Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high selectivity and affinity. The past few years have seen a dramatic increase in aptamer development and interest for diagnostic and therapeutic applications. As the applications for aptamers expand, the need for a more standardized, stringent, and informative characterization and validation methodology increases. Here we performed a comprehensive analysis of a panel of conventional affinity binding assays using a suite of aptamers for the small molecule target ochratoxin A (OTA). Our results highlight inconsistency between conventional affinity assays and the need for multiple characterization strategies. To mitigate some of the challenges revealed in our head-to-head comparison of aptamer binding assays, we further developed and evaluated a set of novel strategies that facilitate efficient screening and characterization of aptamers in solution. Finally, we provide a workflow that permits rapid and robust screening, characterization, and functional verification of aptamers thus improving their development and integration into novel applications.

Development of a bead-based aptamer/antibody detection system for C-reactive protein.

A multiplexing bead-based platform provides an approach for the development of assays targeting specific analytes for biomonitoring and biosensing applications. Multi-Analyte Profiling (xMAP) assays typically employ a sandwich-type format using antibodies for the capture and detection of analytes of interest, and the system permits the simultaneous quantitation of multiple targets. In this study, an aptamer/antibody assay for the detection of C-reactive protein (CRP) was developed. CRP is an acute phase marker of inflammation whose elevated basal levels are correlated with an increased risk for a number of pathologies. For this assay, an RNA aptamer that binds CRP was conjugated to beads to act as the capture agent. Biotinylated anti-CRP antibody coupled to fluorescently labeled streptavidin was used for quantification of CRP. The detection limit of the CRP assay was 0.4 mg/L in diluted serum. The assay was then used to detect spiked CRP samples in the range of 0.4 to 10mg/L in diluted serum with acceptable recoveries (extrapolated values of 70-130%), including that of a certified reference material (129% recovery). The successful incorporation of the CRP aptamer into this platform demonstrates that the exploration of other aptamer-target systems could increase the number of analytes measurable using xMAP-type assays.

An in solution assay for interrogation of affinity and rational minimer design for small molecule-binding aptamers.

Aptamers are short single-stranded oligonucleotides that fold into unique three-dimensional structures, facilitating selective and high affinity binding to their cognate targets. It is not well understood how aptamer-target interactions affect regions of structure in an aptamer, particularly for small molecule targets where binding is often not accompanied by a dramatic change in structure. The DNase I footprinting assay is a classical molecular biology technique for studying DNA-protein interactions. The simplest application of this method permits identification of protein binding where DNase I digestion is inhibited. Here, we describe a novel variation of the classical DNase I assay to study aptamer-small molecule interactions. Given that DNase I preferentially cleaves duplex DNA over single-stranded DNA, we are able to identify regions of aptamer structure that are affected by small molecule target binding. Importantly, our method allows us to quantify these subtle effects, providing an in solution measurement of aptamer-target affinity. We applied this method to study aptamers that bind to the mycotoxin fumonisin B1, allowing the first identification of high affinity putative minimers for this important food contaminant. We confirmed the binding affinity of these minimers using a magnetic bead binding assay.

High resolution grating-assisted surface plasmon resonance fiber optic aptasensor.

A surface plasmon resonance biochemical sensor based on a tilted fiber Bragg grating imprinted in a single mode fiber core is demonstrated. A 30-50 nm thick gold coating on the cladding of the fiber provides the support for surface plasmon waves whose interaction with attached biomolecules is monitored at near infrared wavelengths near 1,550 nm. The transmission spectrum of the sensor provides a fine comb of narrowband resonances that overlap with the broader absorption of the surface plasmon and thus provide a unique tool to measure small shifts of the plasmon with high accuracy. The attachment on the gold surfaces of aptamers with specific affinities for proteins provides the required target-analyte system and is shown to be functional in the framework of our sensing device. The implementation of the sensor either as a stand-alone device or as part of a multi-sensor platform is also described.

Smart materials based on DNA aptamers: taking aptasensing to the next level.

"Smart" materials are an emerging category of multifunctional materials with physical or chemical properties that can be controllably altered in response to an external stimulus. By combining the standard properties of the advanced material with the unique ability to recognize and adapt in response to a change in their environment, these materials are finding applications in areas such as sensing and drug delivery. While the majority of these materials are responsive to physical or chemical changes, a particularly exciting area of research seeks to develop smart materials that are sensitive to specific molecular or biomolecular stimuli. These systems require the integration of a molecular recognition probe specific to the target molecule of interest. The ease of synthesis and labeling, low cost, and stability of DNA aptamers make them uniquely suited to effectively serve as molecular recognition probes in novel smart material systems. This review will highlight current work in the area of aptamer-based smart materials and prospects for their future applications.

Non-invasive Monitoring of α-Synuclein in Saliva for Parkinson's Disease Using Organic Electrolyte-Gated FET Aptasensor.

Parkinson's disease (PD) currently affects more than 1 million people in the US alone, with nearly 8.5 million suffering from the disease worldwide, as per the World Health Organization. However, there remains no fast, pain-free, and effective method of screening for the disease in the ageing population, which also happens to be the most susceptible to this neurodegenerative disease. αSynuclein (αSyn) is a promising PD biomarker, demonstrating clear delineations between levels of the αSyn monomer and the extent of αSyn aggregation in the saliva of PD patients and healthy controls. In this work, we have demonstrated a laboratory prototype of a soft fluidics integrated organic electrolyte-gated field-effect transistor (OEGFET) aptasensor platform capable of quantifying levels of αSyn aggregation in saliva. The aptasensor relies on a recently reported synthetic aptamer which selectively binds to αSyn monomer as the bio-recognition molecule within the integrated fluidic channel of the biosensor. The produced saliva sensor is label-free, fast, and reusable, demonstrating good selectivity only to the target molecule in its monomer form. The novelty of these devices is the fully isolated organic semiconductor, which extends the shelf life, and the novel fully integrated soft microfluidic channels, which simplify saliva loading and testing. The OEGFET aptasensor has a limit of detection of 10 fg/L for the αSyn monomer in spiked saliva supernatant solutions, with a linear range of 100 fg/L to 10 µg/L. The linear range covers the physiological range of the αSyn monomer in the saliva of PD patients. Our biosensors demonstrate a desirably low limit of detection, an extended linear range, and fully integrated microchannels for saliva sample handling, making them a promising platform for non-invasive point-of-care testing of PD.

Aptamer-based colorimetric and lateral flow assay approaches for the detection of toxic metal ions, thallium(i) and lead(ii).

Thallium(i) and lead(ii) ions are heavy metals and extremely toxic. These metals are environmental pollutants, posing a severe risk to the environment and human health. In this study, two approaches were examined using aptamer and nanomaterial-based conjugates for thallium and lead detection. The first approach utilized an in-solution adsorption-desorption approach to develop colorimetric aptasensors for the detection of thallium(i) and lead(ii) using gold or silver nanoparticles. The second approach was the development of lateral flow assays, and their performance was tested with thallium (limit of detection is 7.4 µM) and lead ion (limit of detection is 6.6 nM) spiked into real samples. The approaches assessed are rapid, inexpensive, and time efficient with the potential to become the basis for future biosensor devices.

Computational approaches toward the design of pools for the in vitro selection of complex aptamers.

It is well known that using random RNA/DNA sequences for SELEX experiments will generally yield low-complexity structures. Early experimental results suggest that having a structurally diverse library, which, for instance, includes high-order junctions, may prove useful in finding new functional motifs. Here, we develop two computational methods to generate sequences that exhibit higher structural complexity and can be used to increase the overall structural diversity of initial pools for in vitro selection experiments. Random Filtering selectively increases the number of five-way junctions in RNA/DNA pools, and Genetic Filtering designs RNA/DNA pools to a specified structure distribution, whether uniform or otherwise. We show that using our computationally designed DNA pool greatly improves access to highly complex sequence structures for SELEX experiments (without losing our ability to select for common one-way and two-way junction sequences).