Assessing the clinical utility of biomarkers using the intervention probability curve (IPC).

BACKGROUND: Assessing the clinical utility of biomarkers is a critical step before clinical implementation. The reclassification of patients across clinically relevant subgroups is considered one of the best methods to estimate clinical utility. However, there are important limitations with this methodology. We recently proposed the intervention probability curve (IPC) which models the likelihood that a provider will choose an intervention as a continuous function of the probability, or risk, of disease. OBJECTIVE: To assess the potential impact of a new biomarker for lung cancer using the IPC. METHODS: The IPC derived from the National Lung Screening Trial was used to assess the potential clinical utility of a biomarker for suspected lung cancer. The summary statistics of the change in likelihood of intervention over the population can be interpreted as the expected clinical impact of the added biomarker. RESULTS: The IPC analysis of the novel biomarker estimated that 8% of the benign nodules could avoid an invasive procedure while the cancer nodules would largely remain unchanged (0.1%). We showed the benefits of this approach compared to traditional reclassification methods based on thresholds. CONCLUSIONS: The IPC methodology can be a valuable tool for assessing biomarkers prior to clinical implementation.

Improving lung cancer diagnosis with cancer, fungal, and imaging biomarkers.

OBJECTIVE: Indeterminate pulmonary nodules (IPNs) represent a significant diagnostic burden in health care. We aimed to compare a combination clinical prediction model (Mayo Clinic model), fungal (histoplasmosis serology), imaging (computed tomography [CT] radiomics), and cancer (high-sensitivity cytokeratin fraction 21; hsCYFRA 21-1) biomarker approach to a validated prediction model in diagnosing lung cancer. METHODS: A prospective specimen collection, retrospective blinded evaluation study was performed in 3 independent cohorts with 6- to 30-mm IPNs (n = 281). Serum histoplasmosis immunoglobulin G and immunoglobulin M antibodies and hsCYFRA 21-1 levels were measured and a validated CT radiomic score was calculated. Multivariable logistic regression models were estimated with Mayo Clinic model variables, histoplasmosis antibody levels, CT radiomic score, and hsCYFRA 21-1. Diagnostic performance of the combination model was compared with that of the Mayo Clinic model. Bias-corrected clinical net reclassification index (cNRI) was used to estimate the clinical utility of a combination biomarker approach. RESULTS: A total of 281 patients were included (111 from a histoplasmosis-endemic region). The combination biomarker model including the Mayo Clinic model score, histoplasmosis antibody levels, radiomics, and hsCYFRA 21-1 level showed improved diagnostic accuracy for IPNs compared with the Mayo Clinic model alone with an area under the receiver operating characteristics curve of 0.80 (95% CI, 0.76-0.84) versus 0.72 (95% CI, 0.66-0.78). Use of this combination model correctly reclassified intermediate risk IPNs into low- or high-risk category (cNRI benign = 0.11 and cNRI malignant = 0.16). CONCLUSIONS: The addition of cancer, fungal, and imaging biomarkers improves the diagnostic accuracy for IPNs. Integrating a combination biomarker approach into the diagnostic algorithm of IPNs might decrease unnecessary invasive testing of benign nodules and reduce time to diagnosis for cancer.

Assay Performance of a Label-Free, Solution-Phase CYFRA 21-1 Determination.

CYFRA 21.1, a cytokeratin fragment of epithelial origin, has long been a valuable blood-based biomarker. As with most biomarkers, the clinical diagnostic value of CYFRA 21.1 is dependent on the quantitative performance of the assay. Looking toward translation, it is shown here that a free-solution assay (FSA) coupled with a compensated interferometric reader (CIR) can be used to provide excellent analytical performance in quantifying CYFRA 21.1 in patient serum samples. This report focuses on the analytical performance of the high-sensitivity (hs)-CYFRA 21.1 assay in the context of quantifying the biomarker in two indeterminate pulmonary nodule (IPN) patient cohorts totaling 179 patients. Each of the ten assay calibrations consisted of 6 concentrations, each run as 7 replicates (e.g., 10 × 6 × 7 data points) and were performed on two different instruments by two different operators. Coefficients of variation (CVs) for the hs-CYFRA 21.1 analytical figures of merit, limit of quantification (LOQ) of ca. 60 pg/mL, B max, initial slope, probe-target binding affinity, and reproducibility of quantifying an unknown were found to range from 2.5 to 8.3%. Our results demonstrate the excellent performance of our FSA-CIR hs-CYFRA 21-1 assay and a proof of concept for potentially redefining the performance characteristics of this existing important candidate biomarker.

Integrated Biomarkers for the Management of Indeterminate Pulmonary Nodules.

Rationale: Patients with indeterminate pulmonary nodules (IPNs) at risk of cancer undergo high rates of invasive, costly, and morbid procedures. Objectives: To train and externally validate a risk prediction model that combined clinical, blood, and imaging biomarkers to improve the noninvasive management of IPNs. Methods: In this prospectively collected, retrospective blinded evaluation study, probability of cancer was calculated for 456 patient nodules using the Mayo Clinic model, and patients were categorized into low-, intermediate-, and high-risk groups. A combined biomarker model (CBM) including clinical variables, serum high sensitivity CYFRA 21-1 level, and a radiomic signature was trained in cohort 1 (n = 170) and validated in cohorts 2-4 (total n = 286). All patients were pooled to recalibrate the model for clinical implementation. The clinical utility of the CBM compared with current clinical care was evaluated in 2 cohorts. Measurements and Main Results: The CBM provided improved diagnostic accuracy over the Mayo Clinic model with an improvement in area under the curve of 0.124 (95% bootstrap confidence interval, 0.091-0.156; P < 2 × 10-16). Applying 10% and 70% risk thresholds resulted in a bias-corrected clinical reclassification index for cases and control subjects of 0.15 and 0.12, respectively. A clinical utility analysis of patient medical records estimated that a CBM-guided strategy would have reduced invasive procedures from 62.9% to 50.6% in the intermediate-risk benign population and shortened the median time to diagnosis of cancer from 60 to 21 days in intermediate-risk cancers. Conclusions: Integration of clinical, blood, and image biomarkers improves noninvasive diagnosis of patients with IPNs, potentially reducing the rate of unnecessary invasive procedures while shortening the time to diagnosis.

Correction to Free Solution Assay Signal Modulation in Variable-Stem-Length Hairpin Aptamers.

[This corrects the article DOI: 10.1021/acsomega.9b04341.].

Unlabeled lysophosphatidic acid receptor binding in free solution as determined by a compensated interferometric reader.

Native interactions between lysophospholipids (LPs) and their cognate LP receptors are difficult to measure because of lipophilicity and/or the adhesive properties of lipids, which contribute to high levels of nonspecific binding in cell membrane preparations. Here, we report development of a free-solution assay (FSA) where label-free LPs bind to their cognate G protein-coupled receptors (GPCRs), combined with a recently reported compensated interferometric reader (CIR) to quantify native binding interactions between receptors and ligands. As a test case, the binding parameters between lysophosphatidic acid (LPA) receptor 1 (LPA1; one of six cognate LPA GPCRs) and LPA were determined. FSA-CIR detected specific binding through the simultaneous real-time comparison of bound versus unbound species by measuring the change in the solution dipole moment produced by binding-induced conformational and/or hydration changes. FSA-CIR identified KD values for chemically distinct LPA species binding to human LPA1 and required only a few nanograms of protein: 1-oleoyl (18:1; KD = 2.08 ± 1.32 nM), 1-linoleoyl (18:2; KD = 2.83 ± 1.64 nM), 1-arachidonoyl (20:4; KD = 2.59 ± 0.481 nM), and 1-palmitoyl (16:0; KD = 1.69 ± 0.1 nM) LPA. These KD values compared favorably to those obtained using the previous generation back-scattering interferometry system, a chip-based technique with low-throughput and temperature sensitivity. In conclusion, FSA-CIR offers a new increased-throughput approach to assess quantitatively label-free lipid ligand-receptor binding, including nonactivating antagonist binding, under near-native conditions.

Free Solution Assay Signal Modulation in Variable-Stem-Length Hairpin Aptamers.

Interferometric measurements of free solution assays (FSAs) quantify changes in molecular conformation and hydration upon binding. Here, we demonstrate that aptamer probes designed to undergo varying levels of conformational change upon binding produce corresponding variations in FSA signals. A series of hairpin aptamers were synthesized for the small molecule (tenofovir) with identical loop regions that contain the binding pocket, with between 2 and 10 self-associating base pairings in the stem region. Aptamers selected for tenofovir showed a decrease in the FSA signal and binding affinity (increase in K D) with increasing stem length. Thermodynamic calculations of the Gibbs free energy (ΔG) reported a decrease in ΔG with respect to a corresponding increase in the aptamer stem length. Collectively, these observations provide an expanded understanding of FSA and demonstrate the potential for the rational design of label-free aptamer beacons using FSA as readout.

Quantification of Opioids in Urine Using an Aptamer-Based Free-Solution Assay.

The opioid epidemic continues in the United States. Many have been impacted by this epidemic, including neonates who exhibit Neonatal Abstinence Syndrome (NAS). Opioid diagnosis and NAS can be negatively impacted by limited testing options outside the hospital, due to poor assay performance, false-negatives, rapid drug clearance rates, and difficulty in obtaining enough specimen for testing. Here we report a small volume urine assay for oxycodone, hydrocodone, fentanyl, noroxycodone, norhydrocodone, and norfentanyl with excellent LODs and LOQs. The free-solution assay (FSA), coupled with high affinity DNA aptamer probes and a compensated interferometric reader (CIR), represents a potential solution for quantifying opioids rapidly, at high sensitivity, and noninvasively on small sample volumes. The mix-and-read test is 5- to 275-fold and 50- to 1250-fold more sensitive than LC-MS/MS and immunoassays, respectively. Using FSA, oxycodone, hydrocodone, fentanyl, and their urinary metabolites were quantified using 10 µL of urine at 28-81 pg/mL, with >95% specificity and excellent accuracy in ∼1 h. The assay sensitivity, small sample size requirement, and speed could enable opioid screening, particularly for neonates, and points to the potential for pharmacokinetic tracking.

Compensated Interferometry Measures of CYFRA 21-1 Improve Diagnosis of Lung Cancer.

Diagnosis of lung cancer patients with indeterminate pulmonary nodules (IPNs) presents a significant clinical challenge, with morbidity and management costs of $28 billion/year. We show that a quantitative free-solution assay (FSA), coupled with a compensated interferometric reader (CIR), improves the diagnostic performance of CYFRA 21-1 as a lung cancer biomarker. FSA-CIR is a rapid, mix-and-read, isothermal, label- and enzyme-free, matrix-insensitive, and target and probe-agnostic assay. Operating FSA-CIR at ∼40, 0.75 µL samples/day delivered a serum CYFRA 21-1 limit of quantification (LOQ) of 81 pg/mL with intra-assay and interassay CVs of 4.9% and 9.6% for four-day replicate determinations. Blinded analysis of a 225 patient cohort, consisting of 75 nonmalignant nodules, 45 adenocarcinomas, 44 squamous cell carcinomas, and 61 small cell lung cancers, gave a clear separation of cases and controls, not observed in the Cobas ECL analysis. The area under the curve (AUC) for the Mayo model increased from 0.595 to 0.923 when combined with the FSA-CIR CYFRA 21-1 measurements. In a population with nodules between 6 and 30 mm, the AUC increased from 0.567 to 0.943. In this subgroup, the positive predictive value (PPV) for all tumors by the CYFRA 21-1 assay was 98.7%. Our results demonstrate increased performance of the CYFRA 21-1 assay using FSA-CIR and represents a proof of concept for redefining the performance characteristics of this important candidate biomarker.

Rapid quantification of two chemical nerve agent metabolites in serum.

Organophosphorus compounds (OPs) continue to represent a significant chemical threat to humans due to exposures from their use as weapons, their potential storage hazards, and from their continued use agriculturally. Existing methods for detection include ELISA and mass spectrometry. The new approach presented here provides an innovative first step toward a portable OP quantification method that surmounts conventional limitations involving sensitivity, selectivity, complexity, and portability. DNA affinity probes, or aptamers, represent an emerging technology that, when combined with a mix-and-read, free-solution assay (FSA) and a compensated interferometer (CI) can provide a novel alternative to existing OP nerve agent (OPNA) quantification methods. Here it is shown that FSA can be used to rapidly screen prospective aptamers in the biological matrix of interest, allowing the identification of a 'best-in-class' probe. It is also shown that combining aptamers with FSA-CI enables quantification of the OPNA metabolites, Sarin (NATO designation "G-series, B", or GB) and Venomous Agent X (VX) acids, rapidly with high selectivity at detection limits of sub-10 pg/mL in 25% serum (by volume in PBS). These results suggest there is potential to directly impact diagnostic specificity and sensitivity of emergency response testing methods by both simplifying sample preparation procedures and making a benchtop reader available for OPNA metabolite quantification.

Lysophospholipid G protein-coupled receptor binding parameters as determined by backscattering interferometry.

Lysophosphatidic acid (LPA) activates cognate G protein-coupled receptors (GPCRs) to initiate biological signaling cascades. Lysophospholipid (LP) receptor binding properties remain incompletely assessed because of difficulties with ligand lipophilicity and lipid "stickiness." These inherent attributes produce high levels of nonspecific binding within cell-membrane preparations used to assess GPCRs, as has been shown in classical binding assays using radiolabeled ligands, making accurate measurements of lipid binding kinetics difficult to achieve. Backscattering interferometry (BSI) is an optical technology that measures molecular binding interactions by reporting changes in the refractive index of a solution after binding events. Here, we report the use of BSI to assess LPA1 for its ability to bind to naturally occurring lipids and a synthetic LPA1 antagonist (ONO-9780307), under both primary- and competition-binding conditions. Assessment of 12 different lipids demonstrated that the known LP ligand, 1-oleoyl-LPA, as well as an endocannabinoid metabolite, anandamide phosphate, are specific ligands for LPA1, whereas other LPs tested were not. Newly determined dissociation constants (Kd values) for orthosteric lipid ligands approximated 10-9 M, substantially lower (i.e., with higher affinity) than measured Kd values in classical binding or cell-based assays. These results demonstrate that BSI may have particular utility in assessing binding interactions between lipid receptors and their lipid ligands and could provide new screening approaches for lipid receptor identification and drug discovery.

A Highly Compensated Interferometer for Biochemical Analysis.

Here we report an improved interferometric sensing approach that facilitates high sensitivity nanovolume refractive index (RI) measurements and molecular interaction assays without a temperature controller. The compensated backscattering interferometer (CBSI) is based on a helium-neon (He-Ne) laser, a microfluidic chip, and a CCD array. The CBSI enables simultaneous differential RI measurements within nanoliter volumes, at a compensation level of ca. 5 × 10-8 RIU in the presence of large thermal perturbations (8 °C). This level of d n/d T compensation is enabled by elongating the laser beam along the central axis of the microfluidic channel and measuring the difference in positional shift of interference patterns from two adjacent regions of the channel. By separating two solutions by an air gap or oil droplet, CBSI can discriminate the difference in RI for the sample and reference at a detection limit of 7 × 10-7 RIU in the absence of electronic filtering. At this level of ΔRI sensitivity, it is possible to perform label-free, free-solution biochemical assays at the 10s of nM level without the typical high-resolution temperature control needed in conventional interferometers. Here we illustrate the effective use of CBSI by quantifying the binding affinities for mannose-concanavalin A and Ca2+-recoverin interactions.

Longitudinal pixel averaging for improved compensation in backscattering interferometry.

Longitudinal averaging of the interference pattern in a compensated backscattering interferometer provides improved compensation for temperature induced refractive index perturbations. Fringe pattern likeness between two discrete detection regions of an off-the-shelf microfluidic chip illuminated by an inexpensive diode laser scales with interrogation length. Averaging the intensity distribution along a 2.75 mm length of the channel results in a 750-fold reduction in sensitivity to temperature and a baseline noise level of 3×10-8 refractive index units (RIU). These observations enable nanoliter-volume interferometric measurements at a level of 10-7 RIU in the presence of a 2°C temperature variation without the need for temperature control.

Metallo-ß-lactamase Domain-Containing Protein 1 (MBLAC1) Is a Specific, High-Affinity Target for the Glutamate Transporter Inducer Ceftriaxone.

Ceftriaxone, a ß-lactam antibiotic, has been reported to act independently of its antimicrobial actions to normalize perturbed central nervous system glutamate levels, principally by elevating expression of glial glutamate transporters. Identification of a specific, high-affinity target for ceftriaxone could significantly impact therapeutic development for multiple brain disorders, ranging from neurodegenerative disorders to addiction. Recently, we identified a glial-expressed Caenorhabditis elegans gene, swip-10, that encodes a metallo-ß-lactamase domain-containing protein, and limits glutamate-dependent changes in dopamine neuron excitability. Bioinformatic analyses identified MBLAC1 as the likely mammalian orthologue of swip-10. Using cyanogen bromide immobilized ceftriaxone for affinity capture experiments and backscattering interferometry to monitor MBLAC1 binding of unmodified ceftriaxone, we obtained evidence for specific, high affinity (KD = 2.2 µM) binding of ceftriaxone to MBLAC1. We discuss our findings with respect to MBLAC1 as a potentially exclusive, high-affinity binding partner of ceftriaxone in the CNS, and the path forward in the development of novel, MBLAC1-based therapeutics.

Label-free quantification of calcium-sensor targeting to photoreceptor guanylate cyclase and rhodopsin kinase by backscattering interferometry.

Quantification of protein binding to membrane proteins is challenging and a limited set of methods is available to study such systems. Here we employed backscattering interferometry (BSI), a free-solution label-free method with high sensitivity, to quantify the interaction of neuronal Ca2+-Sensor proteins with their targets operating in phototransduction. We tested direct binding of guanylate cyclase-activating proteins (GCAP1 and GCAP2) to their membrane target guanylate cyclase 1. The regulatory mechanism of GCAPs including their binding interface in the target is unresolved. Here we used a label-free, free-solution assay method based on BSI to determine binding constants of GCAP1 and GCAP2 to the full-length membrane-bound guanylate cyclase type 1. GCAP1 and GCAP2 bound to different regions on the target guanylate cyclase with submicromolar affinity (apparent KD-values of 663 ± 121 nM and 231 ± 63 nM for Ca2+-free GCAP1 and GCAP2, respectively). A guanylate cyclase construct containing the juxta-membrane and kinase homology domain harbored an exclusive binding site for GCAP1 with similar affinities as the full-length protein, whereas GCAP2 did not bind to this region. We provide a model in which GCAP1 and GCAP2 do not share a single binding site to the target, thus cannot exchange upon fluctuating Ca2+ levels.

Physiologically relevant binding affinity quantification of monoclonal antibody PF-00547659 to mucosal addressin cell adhesion molecule for in vitro in vivo correlation.

BACKGROUND AND PURPOSE: A monoclonal antibody (PF-00547659) against mucosal addressin cell adhesion molecule (MAdCAM), expressed as both soluble (sMAdCAM) and trans-membrane (mMAdCAM) target forms, showed over 30-fold difference in antibody-target KD between in vitro (Biacore) and clinically derived (KD,in-vivo ) values. Back-scattering interferometry (BSI) was applied to acquire physiologically relevant KD values which were used to establish in vitro and in vivo correlation (IVIVC). EXPERIMENTAL APPROACH: BSI was applied to obtain KD values between PF-00547659 and recombinant human MAdCAM in buffer or CHO cells and endogenous MAdCAM in human serum or colon tissue. CHO cells and tissue were minimally processed to yield homogenate containing membrane vesicles and soluble proteins. A series of binding affinities in serum with various dilution factors was used to estimate both KD,in-vivo and target concentrations; MAdCAM concentrations were also measured using LC-MS/MS. KEY RESULTS: BSI measurements revealed low KD values (higher affinity) for sMAdCAM in buffer and serum, yet a 20-fold higher KD value (lower affinity) for mMAdCAM in CHO, mMAdCAM and sMAdCAM in tissue. BSI predicted KD,in-vivo in serum was similar to clinically derived KD,in-vivo , and the BSI-estimated serum sMAdCAM concentration also matched the measured concentration by LC-MS/MS. CONCLUSIONS AND IMPLICATIONS: Our results successfully demonstrated that BSI measurements of physiologically relevant KD values can be used to establish IVIVC, for PF-00547659 to MAdCAM despite the lack of correlation when using Biacore measured KD and accurately estimates endogenous target concentrations. The application of BSI would greatly enhance successful basic pharmacological research and drug development.

Origin and prediction of free-solution interaction studies performed label-free.

Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for free-solution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R(2)> 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution.

Quantification of Plasmodium-host protein interactions on intact, unmodified erythrocytes by back-scattering interferometry.

BACKGROUND: Invasion of host erythrocytes by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion involves recognition events between erythrocyte receptors and ligands on the merozoite, the invasive blood form of the parasite. Identifying and characterizing host-parasite interactions is impeded by the biochemical challenges of working with membrane-embedded glycoprotein receptors. For example, the interaction between P. falciparum erythrocyte binding antigen 175 (PfEBA175) and glycophorin A (GYPA) depends on post-translational modifications that are not easily added in recombinant expression systems, and the use of native GYPA is limited by the hydrophobic transmembrane region, making it difficult to biochemically manipulate. It would, therefore, be desirable to perform quantitative binding assays with receptors embedded within the membranes of intact human erythrocytes. METHODS: The extracellular region of GYPA was over-expressed as a soluble protein in HEK293E cells. This protein was characterized, sialylated and evaluated for binding to the PfEBA175 protein. The label-free and free-solution assay, backscattering interferometry (BSI), was used to perform binding assays of two well-characterized P. falciparum invasion ligands to intact unmodified human erythrocytes. RESULTS: Findings indicate that the post-translational modifications present on native GYPA are required for it to bind recombinant PfEBA175 and that these modifications cannot be recapitulated in vitro using mammalian overexpression methods. Here, BSI was used to obtain quantitative, high fidelity interaction determinations on intact, unmodified erythrocytes. Using BSI and purified recombinant proteins constituting the entire ectodomains of the P. falciparum merozoite ligands PfEBA175 and PfRH5, K Ds of 1.1 µM and 50 nM were measured for the PfRH5-BSG and PfEBA175-GYPA interactions, respectively, in good agreement with previous biophysical measurements of these interactions. CONCLUSIONS: These results demonstrate that BSI can be used to detect and quantify the interactions of two merozoite invasion ligands with their receptors on intact human erythrocytes. BSI assays were performed on unlabelled, free-solution proteins in their native environment, requiring only nanomoles of recombinant protein. This study suggests that BSI can be used to investigate host-parasite protein interactions without the limitations of other assay platforms, and therefore represents a valuable new method to investigate the molecular mechanisms involved in erythrocyte invasion by P. falciparum.

Characterizing aptamer small molecule interactions with backscattering interferometry.

Aptamers are segments of single-strand DNA or RNA used in a wide array of applications, including sensors, therapeutics, and cellular process regulators. Aptamers can bind many target species, including proteins, peptides, and small molecules (SM) with high affinity and specificity. They are advantageous because they can be identified in vitro by SELEX, produced rapidly and relatively economically using oligonucleotide synthesis. The use of aptamers as SM probes has experienced a recent rebirth, and because of their unique properties they represent an attractive alternative to antibodies. Current assay methodology for characterizing small molecule-aptamer binding is limited by either mass sensitivity, as in biolayer interferometry (BLI) and surface plasmon resonance (SPR), or the need for using a fluorophore, as in thermophoresis. Here we report that backscattering interferometry (BSI), a label-free and free-solution sensing technique, can be used to effectively characterize SM-aptamer interactions, providing Kd values on microliter sample quantities and at low nanomolar sensitivity. To demonstrate this capability we measured the aptamer affinity for three previously reported small molecules; bisphenol A, tenofovir, and epirubicin showing BSI provided values consistent with those published previously. We then quantified the Kd values for aptamers to ampicillin, tetracycline and norepinephrine. All measurements produced R(2) values >0.95 and an excellent signal to noise ratio at target concentrations that enable true Kd values to be obtained. No immobilization or labeling chemistry was needed, expediting the assay which is also insensitive to the large relative mass difference between the interacting molecules.