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According to estimations of the World Health Organization (WHO), there are almost 500M people in the world that suffer from diabetes. Projections suggest this number will surpass 700M by 2045 with global prevalence surpassing 7%. This huge population, alongside people with pre-diabetics, is prone to develop diabetic retinopathy, the leading cause of vision loss in the working age. While early screening can help prevent most cases of vision loss caused by diabetic retinopathy, the vast majority of patients are not being screened periodically as the guidelines instruct. The challenge is to find a reliable and convenient method to screen patients so that efficacy in detection of referral diabetic retinopathy is sufficient while integration with the flow of care is smooth, easy, simple, and cost-efficient. In this research, we described a screening process for more-than-mild retinopathy through the application of artificial intelligence (AI) algorithms on images obtained by a portable, handheld fundus camera. 156 patients were screened for mtmDR indication. Four images were taken per patient, two macula centered and two optic disc centered. The 624 images were taken using the Optomed Aurora fundus camera and were uploaded using Optomed Direct-Upload. Fully blinded and independently, a certified, experienced ophthalmologist (contracted by Optomed and based in Finland) reviewed each patient to determine ground truth. Indications that are different than mtmDR were also documented by the ophthalmologist to meet exclusion criteria. Data was obtained from anonymized images uploaded to the cloud-based AEYE-DS system and analysis results from the AI algorithm were promptly returned to the users. Of the 156 patients, a certified ophthalmologist determined 100% reached sufficient quality of images for grading, and 36 had existing retinal diseases that fall under exclusion criteria, thus, 77% of the participants met the participation criteria. Of the remaining 120 patients, the AEYE-DS system determined that 2 patients had at least one insufficient quality image. AEYE-DS provided readings for each of the 118 remaining patients (98.3% of all patients). These were statistically compared to the output of the ground truth arm. The patient ground truth was defined as the most severe diagnosis from the four patient images; the ophthalmologist diagnosed 54 patients as mtmDR+ (45% prevalence). Of the 54 patients with referable DR, 50 were diagnosed and of the 64 mtmDR- patients, 61 were correctly diagnosed by the AI. In summary, the results of the study in terms of sensitivity and specificity were 92.6% and 95.3%, respectively. The results indicated accurate classification of diabetic patients that required referral to the ophthalmologist and those who did not. The results also demonstrated the potential of efficient screening and easy workflow integration into points of care such as endocrinology clinics.

A rapid test to identify patients with sickle cell disease could have important benefits in low-resource settings. Sickle cell anemia (SCA) affects about 300,000 newborns each year, the majority of whom are born in sub-Saharan Africa. Low-cost therapies are available to treat SCA, but most countries in sub-Saharan Africa lack robust neonatal screening programs needed to identify patients in need of treatment. To address this need, we developed and evaluated a competitive lateral flow assay that identifies patients with SCA (genotype HbSS) in 15 minutes using undiluted whole blood. A small volume of blood (0.5 μL– 3 μL) is mixed with antibody-coated blue latex beads in a tube and applied to the strip. Strips are then placed in a well of running buffer and allowed to run for 10 minutes. Laboratory evaluation with samples containing different proportions of hemoglobin A (HbA) and hemoglobin S (HbS) indicated that the test should enable identification of SCA patients but not persons with sickle cell trait (SCT). We evaluated the test using 41 samples from individuals with SCA, SCT, and normal blood. With visual inspection or quantitative analysis, we found a 98% accuracy when differentiating SCA from normal and SCT samples as a group (90% sensitivity and 100% specificity for identifying SCA). This work demonstrates important steps towards making a lateral flow test for hemoglobinopathies more appropriate for point-of-care use; further work is needed before the test is appropriate for clinical use.

Nucleic acid diagnostics is a growing sector in life sciences. Numerous applications based on the amplification of nucleic acids are utilized to determine the presence, composition, quality, or quantity of the sequence in question. However, amplification is a time-consuming, laborious, and error-prone process. Therefore, it would be preferable to perform tests with a minimized number of amplification cycles or, alternatively, completely without the amplification. We present two very sensitive sandwich assay concepts for a measurement of nucleic acids. The assays are performed either in standard microtitration wells or on microparticles coated with capture probes. Detection-probe-coated europium(III) nanoparticle labels are used for signal generation. The detection limits of the microtitration well and microparticle applications were 4.0 × 105 and 6.1 × 104 copies of target sequence, respectively. The reference assay, based on the detection of europium(III) chelate-labelled detection probes, had a detection limit of 8.5 × 107 copies. Thus, 100–1000-fold improvement in the sensitivity was achieved. The high sensitivity of the designed assays is based on the long-lifetime fluorescence of nanoparticle labels, on time-resolved fluorometry, and on signal rather than target amplification. The presented sandwich assays provide the tool for a development of direct, amplification-free detection of nucleic acids.

Nanoparticle labels conjugated with biomolecules are used in a variety of different assay applications. We investigated the possibility of using europium(III)-labeled 68-nm nanoparticles coated with monoclonal antibodies or streptavidin (SA) to detect prostate-specific antigen (PSA) in serum. The selection of a suitable antibody pair and interference caused by the combination of nanoparticle label and structurally complex analyte were of special interest. A set of antibodies recognizing different epitope areas of PSA was mapped to find the optimal antibody pair for the immunometric nanoparticle-based assay. Different assay configurations were tested to obtain a good correlation with a conventional method based on biotinylated detection antibodies and europium(III) chelate-labeled streptavidin. Monoclonal capture antibody 5E4 was covalently coated on a microtitration well surface; biotinylated 5H6 monoclonal antibody (Mab) was used for detection, and europium(III)-labeled streptavidin-coated nanoparticles were utilized for signal generation. Total PSA concentrations were determined from a panel of male serum samples to test the developed assay. The correlation of the nanoparticle-based and reference assays was good; y=0.9844x−0.1252, R2=0.98, n=27; and the lowest limit of detection of the assay (LLD=0.83 ng/l) was 35-fold lower than for the reference method. The assay application presented here, where a structurally complex analyte is detected, combines the exceptionally high affinity of streptavidin–biotin technology and the high specific activity of long lifetime fluorescence nanoparticle labels. The general characteristics of this combination should permit the development of various immunoassay applications featuring high sensitivity, rapidity, and low consumption of reagents.

The present study describes the development of a quantitative miniaturized single microparticle immunoassay. The main objective of the study was to evaluate the performance of a miniature heterogeneous immunoassay on a single microparticle in respect to assay kinetics, volume, and sensitivity, binding capacity of microparticles and sensitivity using europium(III) nanoparticle labels. The performance of the single microparticle assay of prostate-specific antigen (PSA) was investigated using different-sized microparticles (60–920 μm in diameter) and microtiter well as a solid-phase. Equilibration time of the assay was shown to be dependent in a linear manner on surface-to-volume ratio, i.e. larger surface-to-volume translated to a faster reaction. However, no correlation between PSA binding capacity and equilibration time was observed in these kinetic studies. Only moderate improvement in assay kinetics was found when PSA binding capacity was increased on a microparticle. Using europium(III) nanoparticle labels, 107 nm in diameter, coated with streptavidin a detection sensitivity of 30 ng l −1 (0.1 amol) was achieved in 1 μl total assay volume per microparticle. This was 50-fold higher compared to the same assay performed with intrinsically fluorescent europium(III) labels.

Preparation, characterisation and application of europium(III) chelate-incorporated polystyrene–acrylic acid (AAc) nanoparticle labels featuring diverse AAc proportions is described. Emulsion copolymerisation of styrene and AAc was used to synthesise uniform-sized nanoparticles, approximately 50 nm in diameter. The structural, fluorescence and functional properties of the nanoparticles were characterised to obtain the optimal polymer composition of particulate labels. The AAc content had only a delicate effect on the fluorescence of the chelate and structural characteristics of the particles. The fluorescence spectra or lifetime of the incorporated europium(III) chelate were not notably affected, and all the particles were analogous in size, had monomodal size distributions and good colloidal stability. However, the AAc content affected strongly on the stability of the incorporated dye and functionality of the labels. Nanoparticles having up to 5.4 mass% of AAc were stable and applicable in high-sensitivity assays, where low detection limit and variation were achieved. The nanoparticles possessing 7.2 or 11.5 mass% of AAc lost remarkably the dye content during storage influencing their usability as labels in assays. Overall, the characterisation and employment results achieved evidenced the importance of the structural composition of nanoparticles, moreover, the knowledge of the effects of structural changes is utilisable in the development of improved nanoparticle labels and assay applications.

Preparation and characterization of europium(III), terbium(III), samarium(III), and dysprosium(III) polystyrene nanoparticle labels with lanthanide-specific fluorescence properties has been presented. Emulsion copolymerization of styrene and acrylic acid was used to synthesize uniform-sized nanoparticles approximately 45 nm in diameter. Europium(III) and samarium(III) lanthanides were chelated with 2-naphthoyltrifluoroacetone and trioctylphosphine oxide to dye the spherical particles, whereas terbium(III) and dysprosium(III) chelate complexes contained a newly synthesized ligand, 4-(2,4,6-tridecyloxyphenyl)pyridine-2,6-dicarboxylic acid. The fluorescence properties of the four lanthanides-including a wide Stokes shift, a narrow emission peak, and long fluorescence lifetime-were retained despite the incorporation into the nanoparticles. Furthermore, the nanoparticles, containing more than 1000 lanthanide chelates, were detectable at label concentrations 3 orders of magnitude lower than the corresponding soluble lanthanide chelate labels. The applicability of the labels prepared was demonstrated by a heterogeneous sandwich-type immunoassay for human prostate-specific antigen, where the lowest limits of detection of 1.6, 2.4, 10.1, and 114.2 ng/L were achieved using europium(III), terbium(III), samarium(III), and dysprosium(III) nanoparticles, respectively. The spectral and functional properties of the lanthanide-embedded polystyrene nanoparticles developed here suggest that the technology is applicable for high-sensitivity multicolor assays.

Background : Recent proceedings in utilization of europium(III) chelate-dyed polystyrene nanoparticles as labels have combined the advantages of an enhanced monovalent binding affinity and a high specific activity of nanoparticle–antibody bioconjugate. Our objective was to evaluate the performance of the nanoparticle label technology with biological samples in an immunoassay of free prostate-specific antigen (PSA-F) using a standard microtitration well platform. Methods : Long-lifetime luminescent europium(III)-chelate nanoparticles, 107 nm in diameter, were coated with a PSA-F specific monoclonal antibody. The two-step noncompetitive immunoassay was performed in a microtitration well coated with a second monoclonal antibody. The signal of the surface-bound nanoparticle–antibody bioconjugates was measured directly from the bottom of the well using a standard time-resolved plate fluorometer. Results : The detection limit (mean+2SD) of the nanoparticle-based PSA-F assay was 0.21 ng/l using a 20-μl sample volume. The assay response was linear up to 5 μg/l, and the functional sensitivity was approximately 0.5 ng/l. The within-run imprecision for spiked serum samples at concentrations 0.0005–0.5 μg/l was 6.4–21.8%, and the within-run and between-run imprecisions for serum samples at concentrations 0.2–2.5 μg/l were 3.4–7.2% and 4.4–7.6%, respectively. The concentrations obtained from serum samples correlated well with the reference immunoassay; slope=1.018±0.018; intercept=0.012±0.021 μg/l; S y | x =0.112 μg/l; r =0.993; n =51. Conclusions : The developed method demonstrated acceptable performance characteristics allowing clinical studies utilizing patient samples with extremely low concentrations of PSA-F. The present assay detected PSA-F in most of samples from prostatectomized men and in few samples from healthy women that were nondetectable according to the reference immunoassay.

Long-lifetime fluorescent europium(III) chelate nanoparticles have been shown to be applicable as labels in various heterogeneous and homogeneous immunoassays. Assay performance can be improved when these intrinsically labeled nanoparticles are used in combination with time-resolved fluorescence detection. In several previous publications, we have shown the potential of nanoparticle labels in different assay applications. In heterogeneous assays, the dynamic range of assays at low concentrations can be extended (1)(2)(3). Furthermore, the kinetic characteristics of assays can be improved by use of detection antibody-coated high-specific-activity nanoparticle labels instead of conventionally labeled detection antibodies (4). In homogeneous assays, europium(III) nanoparticles have been shown to be efficient donors in fluorescence resonance energy transfer, enabling simple and rapid high-throughput screening (5). Heterogeneous and homogeneous nanoparticle-label-based assays can be run with various sample matrixes, e.g., serum (3), heparin plasma (3), and mucus (S. Huopalahti, A. Valanne, T. Soukka, R. Vainionpaa, T. Lovgren, and H. Harma, University of Turku, unpublished data). In the present work we describe a kinetic, heterogeneous, one-step immunoassay application in which europium(III) nanoparticle labels are used in an all-in-one dry-reagent format (Aio!™) (6) to produce a simple, rapid, and sensitive sandwich-type assay for thyroid-stimulating hormone (TSH). Conventionally, the Aio! technology is based on the europium(III) chelate label, but in the present …