Ultrasensitive amplification-free detection of circulating miRNA via droplet-based processing of SERS tag-miRNA-magnetic nanoparticle sandwich nanocomplexes on a paper-based electrowetting-on-dielectric platform.

MicroRNAs (miRNAs) have emerged as a promising class of biomarkers for early detection of various cancers, including ovarian cancer. However, quantifying miRNAs in human blood samples is challenging owing to the issues of sensitivity and specificity. In this study, hsa-miR-200a-3p of the miR-200a sub-family, which is a biomarker of ovarian cancer, was used as the analyte to demonstrate the analytical capability of an integrated biosensing platform using an extremely sensitive surface-enhanced Raman scattering (SERS) nanotag-nanoaggregate-embedded beads (NAEBs), magnetic nanoparticles (MNPs), a pair of highly specific locked nucleic acid (LNA) probes, and a semi-automated paper-based electrowetting-on-dielectric (pEWOD) device to provide labor-less and thorough sample cleanup and recovery. A sandwich approach where NAEBs are modified by one LNA-1 probe and MNPs are modified by another LNA-2 probe was applied. Then, the target analyte miRNA-200a-3p was introduced to form a sandwich nanocomplex through hybridization with the pair of LNA probes. The pEWOD device was used to achieve short cleanup time and good recovery of the nanocomplex, bringing the total analysis time to less than 30 min. The detection limit of this approach can reach 0.26 fM through SERS detection. The versatility of this method without the need for RNA extraction from clinical samples is expected to have good potential in detecting other miRNAs.

Ultrasensitive and Rapid Detection of Procalcitonin via Waveguide-Enhanced Nanogold-Linked Immunosorbent Assay for Early Sepsis Diagnosis.

Sepsis, a life-threatening inflammatory response, demands economical, accurate, and rapid detection of biomarkers during the critical "golden hour" to reduce the patient mortality rate. Here, we demonstrate a cost-effective waveguide-enhanced nanogold-linked immunosorbent assay (WENLISA) based on nanoplasmonic waveguide biosensors for the rapid and sensitive detection of procalcitonin (PCT), a sepsis-related inflammatory biomarker. To enhance the limit of detection (LOD), we employed sandwich assays using immobilized capture antibodies and detection antibodies conjugated to gold nanoparticles to bind the target analyte, leading to a significant evanescent wave redistribution and strong nanoplasmonic absorption near the waveguide surface. Experimentally, we detected PCT for a wide linear response range of 0.1 pg/mL to 1 ng/mL with a record-low LOD of 48.7 fg/mL (3.74 fM) in 8 min. Furthermore, WENLISA has successfully identified PCT levels in the blood plasma of patients with sepsis and healthy individuals, offering a promising technology for early sepsis diagnosis.

Ultrasensitive Upconversion Nanoparticle Immunoassay for Human Serum Cardiac Troponin I Detection Achieved with Resonant Waveguide Grating.

Selective detection of biomarkers at low concentrations in blood is crucial for the clinical diagnosis of many diseases but remains challenging. In this work, we aimed to develop an ultrasensitive immunoassay that can detect biomarkers in serum with an attomolar limit of detection (LOD). We proposed a sandwich-type heterogeneous immunosensor in a 3 × 3 well array format by integrating a resonant waveguide grating (RWG) substrate with upconversion nanoparticles (UCNPs). UCNPs were used to label a target biomarker captured by capture antibody molecules immobilized on the surface of the RWG substrate, and the RWG substrate was used to enhance the upconversion luminescence (UCL) of UCNPs through excitation resonance. The LOD of the immunosensor was greatly reduced due to the increased UCL of UCNPs and the reduction of nonspecific adsorption of detection antibody-conjugated UCNPs on the RWG substrate surface by coating the RWG substrate surface with a carboxymethyl dextran layer. The immunosensor exhibited an extremely low LOD [0.24 fg/mL (9.1 aM)] and wide detection range (1 fg/mL to 100 pg/mL) in the detection of cardiac troponin I (cTnI). The cTnI concentrations in human serum samples collected at different times during cyclophosphamide, epirubicin, and 5-fluorouracil (CEF) chemotherapy in a breast cancer patient were measured by an immunosensor, and the results showed that the CEF chemotherapy did cause cardiotoxicity in the patient. Having a higher number of wells in such an array-based biosensor, the sensor can be developed as a high-throughput diagnostic tool for clinically important biomarkers.

Competitive Assay for the Ultrasensitive Detection of Organophosphate Pesticides Based on a Fiber-Optic Particle Plasmon Resonance Biosensor and an Acetylcholinesterase Binding Peptide.

An acetylcholinesterase (AChE) binding-based biosensor was developed for the ultrasensitive detection of organophosphate (OP) pesticides. The biosensor integrates the technique based on fiber-optic particle plasmon resonance detection and a synthetic AChE binding peptide conjugated with gold nanoparticles on the optical fiber surface via an AChE competitive binding assay. The OP pesticides present in the solution hinder the binding of AChE to the peptide on the biosensor by competing for the binding sites present in AChE. The limit of detection obtained for parathion using this method was observed to be 0.66 ppt (2.3 pM). This method shows a wide linear dynamic range of 6 orders. Furthermore, the use of the AChE binding peptide in the biosensor can better discriminate OPs against carbamates by using only a single biosensor. The practical application of this method was tested using spiked samples, which yielded good recovery and reproducibility. The spiked sample required minimal pretreatment before analysis; hence, this biosensor may also be used in the field.

Integration of Power-Free and Self-Contained Microfluidic Chip with Fiber Optic Particle Plasmon Resonance Aptasensor for Rapid Detection of SARS-CoV-2 Nucleocapsid Protein.

The global pandemic of COVID-19 has created an unrivalled need for sensitive and rapid point-of-care testing (POCT) methods for the detection of infectious viruses. For the novel coronavirus SARS-CoV-2, the nucleocapsid protein (N-protein) is one of the most abundant structural proteins of the virus and it serves as a useful diagnostic marker for detection. Herein, we report a fiber optic particle plasmon resonance (FOPPR) biosensor which employed a single-stranded DNA (ssDNA) aptamer as the recognition element to detect the SARS-CoV-2 N-protein in 15 min with a limit of detection (LOD) of 2.8 nM, meeting the acceptable LOD of 106 copies/mL set by the WHO target product profile. The sensor chip is a microfluidic chip based on the balance between the gravitational potential and the capillary force to control fluid loading, thus enabling the power-free auto-flowing function. It also has a risk-free self-contained design to avoid the risk of the virus leaking into the environment. These findings demonstrate the potential for designing a low-cost and robust POCT device towards rapid antigen detection for early screening of SARS-CoV-2 and its related mutants.

Trace Determination of Grouper Nervous Necrosis Virus in Contaminated Larvae and Pond Water Samples Using Label-Free Fiber Optic Nanoplasmonic Biosensor.

We developed a fast (<20 min), label-free fiber optic particle plasmon resonance (FOPPR) immunosensing method to detect nervous necrosis virus (NNV), which often infects high-value economic aquatic species, such as grouper. Using spiked NNV particles in a phosphate buffer as samples, the standard calibration curve obtained was linear (R2 = 0.99) and the limit of detection (LOD) achieved was 2.75 × 104 TCID50/mL, which is superior to that obtained using enzyme-linked immunosorbent assay (ELISA). By using an enhancement method called fiber optic nanogold-linked immunosorbent assay (FONLISA), the LOD can be further improved to <1 TCID50/mL, which is comparable to that found by the conventional qPCR method. Employing the larvae homogenate samples of NNV-infected grouper, the results obtained by the FOPPR biosensor agree with those obtained by the quantitative polymerase chain reaction (qPCR) method. We also examined pond water samples from an infected container in an indoor aquaculture facility. The lowest detectable level of NNV coat protein was found to be 0.17 µg/mL, which is one order lower than the LOD reported by ELISA. Therefore, we demonstrated the potential of the FOPPR biosensor as an outbreak surveillance tool, which is able to give warning indication even when the trend of larvae death toll increment is still not clear.

Ultrasensitive and Rapid Detection of N-Terminal Pro-B-Type Natriuretic Peptide (NT-proBNP) Using Fiber Optic Nanogold-Linked Immunosorbent Assay.

The N-terminal pro-brain natriuretic peptide (NT-proBNP) is considered an important blood biomarker for heart failure. Herein, we report about a fiber optic nanogold-linked immunosorbent assay (FONLISA) method for the rapid, sensitive, and low-cost detection of NT-proBNP. The method is based on a sandwich immunoassay approach that uses two monoclonal NT-proBNP antibodies, a capture antibody (AbC), and a detection antibody (AbD). AbD is conjugated to a free gold nanoparticle (AuNP) to form the free AuNP@AbD conjugate, and AbC is immobilized on an unclad segment of an optical fiber. The detection of analyte (A), in this case NT-proBNP, is based on the signal change due to the formation of an AuNP@AbD-A-AbC complex on the fiber core surface, where a green light transmitted through the optical fiber will decrease in intensity due to light absorption by AuNPs via the localized surface plasmon resonance effect. This method provides a wide linear dynamic range of 0.50~5000 pg·mL-1 and a limit of detection of 0.058 pg·mL-1 for NT-proBNP. Finally, the method exhibits good correlation (r = 0.979) with the commercial central laboratory-based electrochemiluminescent immunoassay method that uses a Roche Cobas e411 instrument. Hence, our method is potentially a suitable tool for point-of-care testing.

Slab waveguide-based particle plasmon resonance optofluidic biosensor for rapid and label-free detection.

An effective bio-sensing platform that would meet the criteria of rapid, simple, and sensitive detection is crucial to translate bench research to clinical applications. However, simultaneously rapid and sensitive biosensing remains challenging for practical biomedical applications. In this study, for the first time, we demonstrate a cost-effective, label-free, real-time, and sensitive slab waveguide-based particle plasmon resonance (WGPPR) biosensor for practical clinical applications. A suspended glass slab waveguide structure with excellent optical confinement properties was designed and fabricated as the biosensor. Gold nanoparticles (AuNPs) were deposited on the top surface of the waveguide layer to significantly enhance the optical near field through the localized surface plasmon resonance (LSPR) effect. When light travels through the waveguide, the change in the local refractive index (RI) near the surface of the AuNPs can be transformed into changes in the intensity of transmitted light, thereby enabling sensitive and real-time detection. The RI sensing experiment shows a good sensor resolution of 1.43 × 10-4 RIU, which represents a 395% enhancement compared to that of the sensor without AuNPs. Through biochemical detection experiments, we measured IgG and determined the detection limit (LOD) at 614 ng mL-1 in ∼4 min, thereby proving the feasibility of the bio-detection sensing functionality. This study demonstrates a new type of WGPPR biosensor, which offers several unique advantages such as simple structure, high sensitivity, and rapid bio-sensing for practical bio-medical sensing applications. The new biosensor also fulfils point-of-care (POC) requirements.

Quantitative and amplification-free detection of SOCS-1 CpG methylation percentage analyses in gastric cancer by fiber optic nanoplasmonic biosensor.

A new innovative approach is essential for early and effective diagnosis of gastric cancer, using promoter hypermethylation of the tumor suppressor, SOCS-1, that is frequently inactivated in human cancers. We have developed an amplification-free fiber optic nanoplasmonic biosensor for detecting DNA methylation of the SOCS-1 human genome. The method is based on the fiber optic nanogold-linked sorbent assay of PCR-free DNA from human gastric tumor tissue and cell lines. We designed a specific DNA probe fabricated on the fiber core surface while the other probe is bioconjugated with gold nanoparticles in free form to allow percentage determination and differentiating the methylated and unmethylated cell lines, further demonstrating the SOCS-1 methylation occurs in cancer patients but not in normal cell lines. The observed detection limit is 0.81 fM for methylated DNA, and the detection time is within 15 min. In addition, our data were significantly correlated to the data obtained from PCR-based pyrosequencing, and yet with superior accuracy. Hence our results provide new insight to the quantitative evaluation of methylation status of the human genome and can act as an alternative to PCR with a great potential.

Dual-functional gold-iron oxide core-satellite hybrid nanoparticles for sensitivity enhancement in biosensors via nanoplasmonic and preconcentration effects.

A common strategy to improve the sensitivity of a biosensor for the detection of a low abundance analyte is to preconcentrate the analyte molecules before detection. A dual-functional gold-iron oxide core-satellite hybrid nanoparticle structure is proposed in this work to overcome the drawbacks of traditional sample pretreatment methods and the methods using non-magnetic nanomaterials for sample pretreatment. The new dual-functional hybrid nanoparticle structure can simultaneously serve as a signal reporter of a biorecognition event and a preconcentrator of a target at an extremely low concentration in a nanoplasmonic biosensor. By utilizing a fiber optic nanogold-linked sorbent assay in the fiber optic particle plasmon resonance (FOPPR) biosensor and an arbitrary DNA sequence as a target, we have demonstrated that the use of the new hybrid nanoparticle structure with magnetic preconcentration improves the limit of detection (LOD) for the DNA by 18 times as compared to the same method without magnetic preconcentration, so that the LOD for detecting the DNA can be as low as 2.6 fM. The new hybrid nanoparticle structure is easy to prepare and its use in the high-sensitivity and low-cost FOPPR biosensor provides vast opportunities in point-of-care applications.

Integrated Graphene Oxide with Noble Metal Nanoparticles to Develop High-Sensitivity Fiber Optic Particle Plasmon Resonance (FOPPR) Biosensor for Biomolecules Determination.

In this research, a direct, simple and ultrasensitive fiber optic particle plasmon resonance (FOPPR) biosensing platform for immunoglobulin G (IgG) detection was developed using a gold nanoparticle/graphene oxide (AuNP/GO) composite as signal amplification element. To obtain the best analytical performance of the sensor, experimental parameters including the surface concentration of GO on the AuNPs, formation time of the GO, the concentration of the anti-IgG and incubation time of anti-IgG were optimized. The calibration plots displayed a good linear relationship between the sensor response (ΔI/I0) and the logarithm of the analyte concentrations over a linear range from 1.0 × 10-10 to 1.0 × 10-6 g/mL of IgG under the optimum conditions. A limit of detection (LOD) of 0.038 ng/mL for IgG was calculated from the standard calibration curve. The plot has a linear relationship (correlation coefficient, R = 0.9990). The analytical performance of present work's biosensor was better than that of our previously reported mixed self-assembled monolayer of 11-mercaptoundecanoic acid/6-mercapto-1-hexanol (MUA/MCH = 1:4) method by about three orders of magnitude. The achieved good sensitivity may be attributed to the synergistic effect between GO and AuNPs in this study. In addition, GO could immobilize more antibodies due to the abundant carboxylic groups on its surface. Furthermore, we also demonstrated that the results from this sensor have good reproducibility, with coefficients of variation (CVs) < 8% for IgG. Therefore, the present strategy provides a novel and convenient method for chemical and biochemical quantification and determination.

MutS protein-based fiber optic particle plasmon resonance biosensor for detecting single nucleotide polymorphisms.

A new biosensing method is presented to detect gene mutation by integrating the MutS protein from bacteria with a fiber optic particle plasmon resonance (FOPPR) sensing system. In this method, the MutS protein is conjugated with gold nanoparticles (AuNPs) deposited on an optical fiber core surface. The target double-stranded DNA containing an A and C mismatched base pair in a sample can be captured by the MutS protein, causing increased absorption of green light launching into the fiber and hence a decrease in transmitted light intensity through the fiber. As the signal change is enhanced through consecutive total internal reflections along the fiber, the limit of detection for an AC mismatch heteroduplex DNA can be as low as 0.49 nM. Because a microfluidic chip is used to contain the optical fiber, the narrow channel width allows an analysis time as short as 15 min. Furthermore, the label-free and real-time nature of the FOPPR sensing system enables determination of binding affinity and kinetics between MutS and single-base mismatched DNA. The method has been validated using a heterozygous PCR sample from a patient to determine the allelic fraction. The obtained allelic fraction of 0.474 reasonably agrees with the expected allelic fraction of 0.5. Therefore, the MutS-functionalized FOPPR sensor may potentially provide a convenient quantitative tool to detect single nucleotide polymorphisms in biological samples with a short analysis time at the point-of-care sites.

Integration of a Thermoelectric Heating Unit with Ionic Wind-Induced Droplet Centrifugation Chip to Develop Miniaturized Concentration Device for Rapid Determination of Salmonella on Food Samples Using Antibody-Functionalized SERS Tags.

When a centrifugation-enriched sample of 100 µL containing the surface-enhanced Raman scattering (SERS) tag-bound bacteria (Salmonella in this study) is siphoned onto a glass slide next to an embedded thermoelectric heating chip, such a sessile droplet is quickly evaporated. As the size of the sample droplet is significantly reduced during the heating process, ionic wind streams from a corona discharge needle, stationed above the sample, sweep across the liquid surface to produce centrifugal vortex flow. Tag-bound Salmonella in the sample are then dragged and trapped at the center of droplet bottom. Finally, when the sample is dried, unlike the "coffee ring" effect, the SERS tag-bound Salmonella is concentrated in one small spot to allow sensitive detection of a Raman signal. Compared with our previous electrohydrodynamic concentration device containing only a corona discharge needle, this thermoelectric evaporation-assisted device is more time-effective, with the time of concentrating and drying about 100 µL sample reduced from 2 h to 30 min. Hence, sample throughput can be accelerated with this device for practical use. It is also more sensitive, with SERS detection of a few cells of Salmonella in neat samples achievable. We also evaluated the feasibility of using this device to detect Salmonella in food samples without performing the culturing procedures. Having spiked a few Salmonella cells into ice cubes and lettuce leaves, we use filtration and ultracentrifugation steps to obtain enriched tag-bound Salmonella samples of 200 µL. After loading an aliquot of 100 µL of sample onto this concentration device, the SERS tag signals from samples of 100 g ice cubes containing two Salmonella cells and 20 g lettuce leaf containing 5 Salmonella cells can be successfully detected.

Label-free SERS characterization of snake venoms by exploring the cysteine environs with bone-shaped gold nanoparticles.

Identification of snake venoms is a vital step in the treatment of fatal snakebites. In this study, we use the gold-thiolate interaction between a cysteine residue and gold nanoparticles to establish a SERS method for the differentiation of the venoms of Trimeresurus stejnegeri and Bungarus multicinctus. We confirm the preference of gold nanoparticles over silver for the SERS study of snake venoms by a binding experiment that also functions to differentiate the two venom samples by colorimetry and UV-vis spectroscopy. We report the SERS spectra of Trimeresurus stejnegeri and Bungarus multicinctus venoms for the first time. The spectra display distinct SERS signatures of the snake venoms on bone-shaped gold nanoparticles made with a house recipe. These signatures correlate to selected segments of the venom proteins due to the anchoring effect of the gold-cysteine bond. The method is quick as it accomplishes in situ isolation of the structure of interest to avoid tedious purification of the samples. The location of the interactive cysteine residue makes a novel characteristic of proteins in general.

Low-cost planar waveguide-based optofluidic sensor for real-time refractive index sensing.

We report on the design, fabrication, and characterization of mass-producible, sensitive, intensity-detection-based planar waveguide sensors for rapid refractive index (RI) sensing; the sensors comprise suspended glass planar waveguides on glass substrates, and are integrated with microfluidic channels. They are facilely and cost-effectively constructed via vacuum-less processes. They yield a high throughput, enabling mass production. The sensors respond to solutions with different RIs via variations in the transmitted optical power due to coupling loss in the sensing region, facilitating real-time and simple RI detection. Experiments yield a good resolution of 5.65 × 10-4 RIU. This work has major implications for several RI-sensing-based applications.

Fiber Optic Particle Plasmon Resonance Biosensor for Label-Free Detection of Nucleic Acids and Its Application to HLA-B27 mRNA Detection in Patients with Ankylosing Spondylitis.

We developed a label-free, real-time, and highly sensitive nucleic acid biosensor based on fiber optic particle plasmon resonance (FOPPR). The biosensor employs a single-strand deoxyoligonucleotides (ssDNA) probe, conjugated to immobilized gold nanoparticles on the core surface of an optical fiber. We explore the steric effects on hybridization affinity and limit of detection (LOD), by using different ssDNA probe designs and surface chemistries, including diluent molecules of different lengths in mixed self-assembled monolayers, ssDNA probes of different oligonucleotide lengths, ssDNA probes in different orientations to accommodate target oligonucleotides with a hybridization region located unevenly in the strand. Based on the optimized ssDNA probe design and surface chemistry, we achieved LOD at sub-nM level, which makes detection of target oligonucleotides as low as 1 fmol possible in the 10-mL sensor chip. Additionally, the FOPPR biosensor shows a good correlation in determining HLA-B27 mRNA, in extracted blood samples from patients with ankylosing spondylitis (AS), with the clinically accepted real-time reverse transcription-polymerase chain reaction (RT-PCR) method. The results from this fundamental study should guide the design of ssDNA probe for anti-sense sensing. Further results through application to HLA-B27 mRNA detection illustrate the feasibility in detecting various nucleic acids of chemical and biological relevance.

A fiber optic nanoplasmonic biosensor for the sensitive detection of ampicillin and its analogs.

A novel optical immunosensor for the screening of ampicillin (Amp) residues has been developed. The biosensor is based on fiber optic particle plasmon resonance detection and uses an enhancement method called as fiber optic nanogold-linked immunosorbent assay (FONLISA) for the sensitive detection of antibiotics. A commercial antibody which had a higher affinity for ampicillin than for other ß-lactam antibiotics was chosen. A surface competitive binding assay was used in which a fixed concentration of antibiotic-conjugated gold nanoparticles (AuNPs) competes with free unlabeled antibiotic molecules to measure the amount of binding with antibody molecules immobilized on an optical fiber. The synthesis of the 11-mercaptoundecanoic acid (MUA)-ampicillin conjugate facilitates the attachment of the Amp molecules to AuNPs via MUA which acts as a linker between them. This AuNP-Amp conjugate was then used for the detection of ß-lactam antibiotics. The practical limit of detection obtained for Amp was 0.74 ppb (7.4 × 10-10 g/mL) which is lower than the recommended maximum residue limit (MRL) for ß-lactams. The method also shows a wide linear range of 4 orders. Its applicability to the determination of ampicillin in spiked milk samples has been demonstrated with good recovery and reproducibility. Graphical abstract.

Controlled Silanization: High Molecular Regularity of Functional Thiol Groups on Siloxane Coatings.

A comparative study on deposition and molecular regularity of two organosilanes, i.e., commercially available (3-mercaptopropyl)trimethoxysilane (MPTMS) and newly developed mercaptopropylsilatrane (MPS), was conducted in this work. MPTMS and MPS were applied to modify silicon surfaces to characterize their deposition kinetics, surface morphology, thickness, and elemental composition and the reactivity of thiol end groups based on gold-thiol and thiol-ene chemistries. MPS possesses a tricyclic caged structure and a transannular N → Si dative bond, making it chemically stable and controllable to avoid fast hydrolysis and aggregation in solution. The results indicate that MPS allows faster deposition and better formation of thin and homogeneous films than MPTMS. More importantly, the functional thiol groups on MPS coatings enable immobilization of a large amount of gold nanoparticles and effective thiol-ene photopolymerization with zwitterionic sulfobetaine acrylamide. Postmodification on silanized surfaces with MPS endows excellent plasmonic and antifouling properties, potentially leading to valuable applications to biosensing and biomaterials. The work demonstrated the feasibility and applicability of the functional silatrane molecule for surface silanization in a controlled manner.

Development of microfluidic concentrator using ion concentration polarization mechanism to assist trapping magnetic nanoparticle-bound miRNA to detect with Raman tags.

MicroRNAs (miRNAs) are small noncoding single-stranded ribonucleic acid molecules. This type of endogenous oligonucleotide could be secreted into the circulation and exist stably. The detection of specific miRNAs released by cancer cells potentially provides a noninvasive means to achieve early diagnosis and prognosis of cancers. However, the typical concentration of miRNAs in blood is below the ultratrace level. This study uses a simple thermoplastic microfluidic concentration device based on an ion concentration polarization mechanism to perform enrichment and cleanup and Raman sensing beads to determine miRNA quantitatively. One sample solution containing target miRNA molecules having been hybridized with two nucleotide probes, where one probe is on a Raman tag of a nanoaggregate embedded bead (NAEB) and the other probe is on a magnetic nanoparticle (MNP), is first filled into the device. When an external field is applied across a cation exchange membrane stationed in the middle conduit of the device, the MNP-miRNA-NAEB complexed particles are enriched near the membrane edge of the cathode side. The concentrated complexed particles are further trapped using an external magnet to perform washing steps to remove excess noncomplexed NAEBs. When cleanup steps are accomplished, the remaining complexed particles are loaded into one detection capillary to acquire Raman signals from the sensing beads. Compared with that using a conventional magnetic trapping device, the cleanup time is shortened from nearly an hour to less than 10 min. Sample loss during the washing steps becomes more controllable, resulting in adequate standard curve linearity (R > 0.99) ranging from 1 to 100 pM.