Spectrally separated dual-label upconversion luminescence lateral flow assay for cancer-specific STn-glycosylation in CA125 and CA15-3.

Multiplexed lateral flow assays (LFAs) offer efficient on-site testing by simultaneously detecting multiple biomarkers from a single sample, reducing costs. In cancer diagnostics, where biomarkers can lack specificity, multiparameter detection provides more information at the point-of-care. Our research focuses on epithelial ovarian cancer (EOC), where STn-glycosylated forms of CA125 and CA15-3 antigens can better discriminate cancer from benign conditions. We have developed a dual-label LFA that detects both CA125-STn and CA15-3-STn within a single anti-STn antibody test line. This utilizes spectral separation of green (540 nm) and blue (450 nm) emitting erbium (NaYF4:Yb3+, Er3+)- and thulium (NaYF4: Yb3+, Tm3+)-doped upconverting nanoparticle (UCNP) reporters conjugated with antibodies against the protein epitopes in CA125 or CA15-3. This technology allows the simultaneous detection of different antigen variants from a single test line. The developed proof-of-concept dual-label LFA was able to distinguish between the ascites fluid samples from diagnosed ovarian cancer patients (n = 10) and liver cirrhosis ascites fluid samples (n = 3) used as a negative control. The analytical sensitivity of CA125-STn for the dual-label LFA was 1.8 U/ml in buffer and 3.6 U/ml in ascites fluid matrix. Here we demonstrate a novel approach of spectrally separated measurement of STn-glycosylated forms of two different cancer-associated protein biomarkers by using UCNP reporter technology.

Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer.

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL-1 (1.5 µg kg-1) and an IC50 value of 0.36 ng mL-1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.

MRI of odontogenic maxillofacial infections: diagnostic accuracy and reliability.

OBJECTIVES: To determine the diagnostic accuracy of emergency magnetic resonance imaging (MRI) in odontogenic maxillofacial infections, the clinical and surgical significance of MRI findings, and whether MRI can identify the tooth responsible for the infection. METHODS: A retrospective cohort study reviewed 106 emergency neck MRI scans of patients with neck infections of odontogenic origin. The diagnostic accuracy of MRI in identifying abscesses was studied relative to surgical findings. Correlations were analyzed between various MRI findings and clinical results and outcomes, such as the surgical approach (intraoral vs. extraoral). The ability of MRI findings to predict the causative tooth was assessed in a blinded multi-reader setting. RESULTS: Of the 106 patients with odontogenic infections, 77 (73%) had one or more abscesses. Imaging showed a sensitivity, specificity, and accuracy of 0.95, 0.84, and 0.92, respectively, for MRI diagnosis of an odontogenic abscess. Among the imaging findings, mediastinal edema was the strongest predictor of extraoral surgery. MRI showed bone marrow edema in the majority of patients, and multi-reader assessment showed good reliability. MRI was also able to predict the causative tooth accurately. CONCLUSIONS: Emergency neck MRI can accurately detect odontogenic abscesses and reliably point to the causative tooth. These results can increase the utility and reliance on emergency MRI in clinical decision-making.

Magnetic resonance imaging findings in pediatric neck infections-a comparison with adult patients.

BACKGROUND: Differences in the functioning of the immune system and the anatomical proportions of the neck between children and adults lead to different manifestations of deep neck infections. Magnetic resonance imaging (MRI) may serve as an alternative to computed tomography (CT) as the primary imaging modality. OBJECTIVE: To study characteristic MRI findings and the diagnostic accuracy of MRI in pediatric deep neck infections. MATERIALS AND METHODS: We retrospectively studied a cohort of pediatric patients who underwent a neck 3-tesla MRI study over a five-year period. Inclusion criteria were: 1) emergency MRI findings indicating an infection, 2) infection as the final clinical diagnosis, 3) diagnostic image quality verified by the radiologist reading the study and 4) age under 18 years. Patient record data, including surgery reports, were compared with the MRI findings. RESULTS: Data of 45 children were included and analysed. Compared to adults, children had a higher incidence of retropharyngeal infection and lymphadenitis, and a lower incidence of peritonsillar/parapharyngeal infection. MRI showed evidence of an abscess in 34 children. Of these 34 patients, 24 underwent surgery, which confirmed an abscess in 21 but no abscess in three patients. In addition, three patients underwent surgery without MRI evidence of abscess, and an abscess was found in one of these cases. The measures of diagnostic accuracy among the children were sensitivity 0.96, specificity 0.77, positive predictive value 0.89, negative predictive value 0.91 and accuracy 0.89. Compared with adults, children had lower C-reactive protein, but a similar proportion of them had an abscess, and abscess size and rate of surgery were similar. CONCLUSION: Despite the differences in the infection foci, emergency MRI in children had equal diagnostic accuracy to that in adults.

Does orthognathic treatment improve patients' psychosocial well-being?

OBJECTIVE: To analyse changes in patients' psychosocial well-being from before treatment until post-surgical orthodontic treatment (including retention) is completed. MATERIALS AND METHODS: Data was collected six times: before treatment (T0), 6-8 weeks after the placement of orthodontic appliances (T2), 3-4 weeks before surgery (T3), six weeks after surgery (T4), one year after surgery (T5) and after completing orthodontic treatment (T6; 20-57 months after surgery). At T0, 60 patients participated while at T6, data was available for 15 patients. All patients completed the Orthognathic Quality of Life Questionnaire (OQLQ), Rosenberg Self-Esteem Questionnaire (RSES), Acceptance and Action Questionnaire II (AAQ-II) and the Symptom Checklist 90 (SCL-90). All pairwise comparisons between variables were conducted with the Wilcoxon signed-rank test. RESULTS: OQLQ function, RSES, AAQ-II and SCL GSI worsened from T0 to T2. At T5, improvements compared to T0 were found in all aspects of OQLQ and SCL GSI. When comparing results at T6 to T0, improvements where only found in OQLQ sum, OQLQ facial aesthetics and OQLQ function. CONCLUSIONS: Although well-being of orthognathic patients seems to improve during treatment, many improvements cannot be verified anymore at the completion of the retention period. Most stable changes are found in the oral function component and in the facial aesthetics component of the OQLQ.

Early surgical intervention enhances recovery of severe pediatric deep neck infection patients.

PURPOSE: Pediatric deep neck space infection (DNI) is a relatively rare but potentially life-threatening condition and requires prompt and accurate management. This study retrospectively reviews our experience in a tertiary referral hospital from 2004 to 2019. METHODS: Systematic data collection from medical records using ICD10 codes between 2004 and 2019. INCLUSION CRITERIA: age ≤16 years, DNI requiring hospitalization and/or surgery. EXCLUSION: peritonsillar abscess without complications. RESULTS: We identified 42 patients, 21 boys and 21 girls, with a median age of 4.9 years. Most of the patients had severe symptoms, the most common of which were neck swelling (n = 39; 92.9%), neck pain (n = 39; 92.9%) and fever (n = 32; 76.2%). Twenty-two (52.4%) had torticollis, and the mean duration of symptoms before hospitalization was 4.95 days. Diagnosis was confirmed by MRI (n = 24), contrast-enhanced CT (n = 11) or ultrasonography (n = 6), except in one case. Twenty-three (54.8%) required an open neck incision, ten (23.8%) patients had intraoral surgery and nine were treated conservatively. Twelve (28.6%) patients were admitted to the pediatric ICU. Median hospitalization duration was six days. The infection most commonly had tonsillopharyngeal etiology (n = 18) and a retropharyngeal location (n = 17). Staphylococcus Aureus (n = 7) and Streptococcus pyogenes (n = 7) were the most frequent pathogens. We compared the early surgical intervention group (<2 days of intravenous antibiotics; n = 18; 42.9%) to the late surgery group (n = 15; 35.7%) and the conservatively treated groups (n = 9; 21.4%). The overall length of stay (LOS) was lower in the shorter preoperative medication group (mean 4.4 vs. 7.2; p = 0.009). The size of the abscess did not differ between the groups (mean 28 mm; 30 mm; 21 mm; p = 0.075) and the neck incision rate was similar in the operated groups. CONCLUSION: Early surgical intervention is associated with decreased LOS among severe pediatric DNI patients.

Gingival cell growth with antiresorptive treatment combined with corticosteroids or antiestrogen.

OBJECTIVES: Antiresorptive treatment has been shown to impair mucosal cell proliferation, migration, and viability. However, in the clinic, antiresorptives are often used in combination with other drugs. We studied the effect of antiresorptives combined with a corticosteroid or antiestrogen on oral mucosal keratinocytes and fibroblasts. MATERIAL AND METHODS: Human gingival keratinocyte and fibroblast cell lines were exposed to bisphosphonates (BPs) and denosumab in different concentrations and durations together with an antiestrogen or corticosteroid. Changes in cell viability, proliferation and migration after exposures were measured. Data were evaluated with hierarchical linear mixed model for repeated measurements. RESULTS: Bisphosphonate exposure suppressed keratinocyte and fibroblast cell viability, proliferation, and migration in a time-dependent manner. Combining a corticosteroid or antiestrogen with BPs further increased this negative effect. Denosumab alone had a mild positive effect on keratinocyte and fibroblast growth. When denosumab was combined with a corticosteroid or antiestrogen, cell growth was suppressed. CONCLUSIONS: Our results show that coexisting medications may increase the negative impact of BPs or denosumab on oral mucosal cells.

Development of a Patient-Centered Functional Outcomes Questionnaire in Head and Neck Cancer.

Importance: Incorporation of patient perspectives, or patient-reported outcomes, in functional outcome measures has been gaining prominence in the literature on reconstructive surgery. Objective: To create and validate an instrument for measuring the main functional areas of concern for patients with head and neck cancer. Design, Setting, and Participants: This 4-phase mixed-methods qualitative study was conducted from July 1, 2013, to June 30, 2016, in a quaternary head and neck oncology center in Edmonton, Alberta, Canada. Patients were recruited from 3 Head and Neck Research Network sites: University of Alberta (Edmonton, Canada), Mount Sinai Health Network (New York, New York), and University of Turku Hospital (Turku, Finland). The inclusion criteria included 18 years of age or older, diagnosis of squamous cell carcinoma involving the subsites of the head and neck (ie, oral cavity, oropharynx, hypopharynx, and larynx), and at least 1 year since treatment completion. Those patients who were undergoing additional active treatment or with evidence of disease recurrence were excluded. Data were analyzed from July 1, 2013, to June 30, 2016. Main Outcomes and Measures: The primary outcome measures were the clinical correlation of the Edmonton-33 instrument scores with swallowing, speech, dry mouth, and chewing assessment outcomes. Results: In total, 10 patients with head and neck cancer (mean age, 59.6 years; 6 men [60%]) were included in phase 1 of the study, 5 patients (mean age, 55.2 years) were included in phase 2, 10 patients were included in phase 3, and 25 patients with head and neck cancer (mean age, 62.6 years; 14 men [56%]) participated in the phase 4 validation. The Edmonton-33 instrument scores correlated strongly with the swallowing scores of the MD Anderson Dysphagia Inventory (r = 0.77; 95% CI, 0.49-1.0), the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Head and Neck 35 (EORTC QLQ-H&N35) (r = -0.73; 95% CI, -1.0 to -0.44), and the modified barium swallow test (r = -0.60; 95% CI, -0.94 to -0.25). The instrument scores were also strongly correlated with the Speech Handicap Index scores (r = -0.64; 95% CI, -0.97 to -0.31), word intelligibility scores (r = 0.61; 95% CI, 0.27-0.95), and sentence intelligibility scores (r = 0.55; 95% CI, 0.19-0.91). A moderate to strong correlation was observed between the Edmonton-33 instrument and the EORTC QLQ-H&N35 scores in the dry mouth (r = -0.54; 95% CI, -0.91 to -0.18) and chewing (r = -0.45; 95% CI, -0.84 to -0.06) domains. The factor loading values for the domains of swallowing, speech, dry mouth, and chewing were all greater than 0.3. The mean factor loading values for the items related to swallowing were 0.71 (95% CI, 0.62-0.80) and for the items related to speech were 0.76 (95% CI, 0.72-0.80). The mean factor loading values for the items related to dry mouth were 0.71 (95% CI, 0.59-0.83) and for those related to chewing were 0.77 (95% CI, 0.69-0.85). Conclusions and Relevance: The Edmonton-33 appears to be a validated instrument that will allow patients with head and neck cancer to assess and report their own functional outcomes. It could serve as a single comprehensive measure for functional outcomes.

Pandemic influenza A(H1N1pdm09) vaccine induced high levels of influenza-specific IgG and IgM antibodies as analyzed by enzyme immunoassay and dual-mode multiplex microarray immunoassay methods.

Influenza A viruses continue to circulate throughout the world as yearly epidemics or occasional pandemics. Influenza infections can be prevented by seasonal multivalent or monovalent pandemic vaccines. In the present study, we describe a novel multiplex microarray immunoassay (MAIA) for simultaneous measurement of virus-specific IgG and IgM antibodies using Pandemrix-vaccinated adult sera collected at day 0 and 28 and 180 days after vaccination as the study material. MAIA showed excellent correlation with a conventional enzyme immunoassay (EIA) in both IgG and IgM anti-influenza A antibodies and good correlation with hemagglutination inhibition (HI) test. Pandemrix vaccine induced 5-30 fold increases in anti-H1N1pdm09 influenza antibodies as measured by HI, EIA or MAIA. A clear increase in virus-specific IgG antibodies was found in 93-97% of vaccinees by MAIA and EIA. Virus-specific IgM antibodies were found in 90-92% of vaccinees by MAIA and EIA, respectively and IgM antibodies persisted for up to 6 months after vaccination in 55-62% of the vaccinees. Pandemic influenza vaccine induced strong anti-influenza A IgG and IgM responses that persisted several months after vaccination. MAIA was demonstrated to be an excellent method for simultaneous measurement of antiviral IgG and IgM antibodies against multiple virus antigens. Thus the method is well suitable for large scale epidemiological and vaccine immunity studies.

Deep neck space infections: an upward trend and changing characteristics.

PURPOSE: This study reviews our experience with deep neck space infections (DNIs) requiring surgical intervention, including cervical necrotizing fasciitis. The aim of the study was to identify predisposing and aggravating factors of the disease and recognize the possible factors that can lead to life-threatening complications and slow down the healing process. METHODS: We compare the results to previous data from 1985 to 2005 to find possible alterations and changing trends. The characteristics of four lethal cases are described. This retrospective analysis includes patient data from 2004 to 2015 in tertiary referral hospital and in total, 277 patients were found. RESULTS: Surgical drainage through a neck opening ± intraoral incision was made in 215 (77.6%) patients, an intraoral incision was only made in 62 patients (22.4%). ICU care was needed in 66 (23.8%) cases. Odontogenic etiology (44.8%) was the most common origin. The most common comorbidity was a psychiatric disorder and/or dementia and occurred in 55 (19.9%) patients. Patients with underlying illnesses were more likely to be admitted to the ICU (p = 0.020), required a longer ICU stay (p = 0.004) and repeated surgery (p = 0.009). Gas formation seemed to be predictive of a more severe course of infection. Early extraction of the odontogenic foci was related to a lower length of stay (LOS) (p = 0.039). CONCLUSION: The annual numbers have risen from 14 to 24 cases per year when compared to previous data. DNIs remain a cause of lethal complications; the mortality was 1.4% and overall complications occurred in 61 (22.0%) patients.

Nanoparticle-aided glycovariant assays to bridge biomarker performance and ctDNA results.

Numerous immunoassay based cancer biomarkers established in the 1970 and 1980'ies are widely used in clinical routine. Initial expectations of biomarkers such as CEA, CA125, CA19-9, AFP to provide decisive help in the diagnosis of early stage, pre-symptomatic cancers have not been realized. Thus, they are primarily used for monitoring disease progression and occasionally being useful as prognostic indicators. This limitation is due to the marker also being measurable in healthy individuals and frequently at elevated concentrations in common benign conditions. Most conventional tumor markers are glycosylated and interestingly specific alterations of the glycostructure part can often be seen early in the cancerous process. Conventional double monoclonal immunoassays are however blind to such changes as they are based on peptide epitope recognition. Wide selections of carbohydrate recognizing macromolecules, lectins, but also glycan structure recognizing antibodies are potentially useful for detecting such changes. Despite numerous attempts generating proof-of-principle evidence for this, such assays have generally not been successfully introduced into clinical routine. The affinity constants of lectin and glycan specific antibodies for their corresponding carbohydrate structures may be up to several orders too low to provide the detection limits and robustness expected from routine tumor markers. In this review, we describe an approach based on the use of highly fluorescent Eu3+--chelate dyed nanoparticles onto which lectins or glycan specific antibodies are coated to provide the necessary binding strength and signal amplification to provide low detection limits, while maintaining the original glycan-structure specificity. This concept applied to three markers, PSA, CA125 and CA15-3 provide glycoform assays of greatly enhanced cancer specificity using sample volumes similar or lower than corresponding traditional ELISAs. For ovarian cancer, we show that this new approach when applied to ovarian cyst fluid samples provide results similar to the performance obtained with ctDNA determinations of a set of 17 driver mutations and greatly superior compared to corresponding conventional immunoassays. Based on our results, we predict that the nanoparticle-lectin concept will enable a new generation of simple, low-cost biomarker assays of highly improved cancer specificity. Such tools should ideally be evaluated together with determination of ctDNA to establish early detection schemes for cancers e.g. ovarian, pancreas, lung where the detection rate of early stage disease is presently unacceptably low.

Serological Array-in-Well Multiplex Assay Reveals a High Rate of Respiratory Virus Infections and Reinfections in Young Children.

Serological assays are used to diagnose and characterize host immune responses against microbial pathogens. Microarray technologies facilitate high-throughput immunoassays of antibody detection against multiple pathogens simultaneously. To improve survey of influenza A virus (IAV), influenza B virus (IBV), respiratory syncytial virus (RSV), and adenovirus (AdV) antibody levels, we developed a microarray consisting of IAV H1N1, IAV H1N1pdm09 (vaccine), IAV H3N2, IBV Victoria, IBV Yamagata, RSV, AdV type 5 hexon protein, and control antigens printed on the bottom of a microtiter plate well. Bound IgG antibodies were detected with anti-human IgG-coated photon-upconverting nanoparticles and measured with a photoluminescence imager. The performance of the microarray immunoassay (MAIA) was evaluated with serum samples (n = 576) collected from children (n = 288) at 1 and 2 years of age and tested by standard enzyme immunoassays (EIAs) for antibodies to IAV vaccine and RSV. EIAs and MAIA showed substantial to almost perfect agreement (Cohen's κ, 0.62 to 0.83). Applying MAIA, we found seroprevalences of 55% for IAV H1N1, 54% for IAV vaccine, 30% for IAV H3N2, 24% for IBV Victoria, 25% for IBV Yamagata, 38% for RSV, and 26% for AdV in 1-year-old children (n = 768). By the age of 2 years, IgG seropositivity rates (n = 714) increased to 74% for IAV H1N1, 71% for IAV vaccine, 49% for IAV H3N2, 47% for IBV Yamagata, 49% for IBV Victoria, 68% for RSV, and 58% for AdV. By analyzing increases in antibody levels not biased by vaccinations, we found a reinfection rate of 40% for RSV and 31% for AdV in children between 1 and 2 years of age.IMPORTANCE The multiplex immunoassay was successfully used to simultaneously detect antibodies against seven different viruses. The developed serological microarray is a new promising tool for diagnostic, epidemiological, and seroprevalence analyses of virus infections.

Benign proliferative epithelial lesions of oral mucosa are infrequently associated with α-, ß-, or γ human papillomaviruses.

BACKGROUND: Oral papillomas and verruca vulgaris have been associated with human papillomavirus (HPV) infection. However, approximately half of these have remained HPV-negative when tested for mucosal HPV genotypes. In this study, we evaluated presence of α-, ß-, and γ-HPVs in benign papillary and verrucous lesions. METHODS: Eighty-three clinical lesions with suspected HPV etiology were analyzed for HPV types of genus α (n = 24), ß (n = 46), and γ (n = 52). Immunohistochemistry was used for p16 as a possible surrogate marker of high-risk HPV, accompanied by Ki-67 proliferation marker. RESULTS: Altogether, α-HPVs were detected in 6.4%, ß-HPVs in 2.4%, and γ-HPV in 4.8%. The following genotypes were identified: HPV6, 8, 11, 16, 22, 161, and 170. Neither Ki-67 nor p16 positivity alone were associated with HPV but combined staining showed significant inverse association (P = .042). CONCLUSION: HPV infection is found only in a minority of benign verrucous and papillary oral lesions, with the predominance of α-HPVs. LEVEL OF EVIDENCE: 4.

Functional and quality of life outcomes after partial glossectomy: a multi-institutional longitudinal study of the head and neck research network.

BACKGROUND: While aggressive treatment for oral cancer may optimize survival, decrements in speech and swallowing function and quality of life often result. This exploratory study investigated how patients recover their communicative function, swallowing ability, and quality of life after primary surgery [with or without adjuvant (chemo)radiation therapy] for tongue cancer over the course of the first year post-operation. METHODS: Patients treated for oral cancer at three institutions (University of Alberta Hospital, Mount Sinai Beth Israel Medical Center, and Turku University Hospital) were administered patient-reported outcomes assessing speech [Speech Handicap Index (SHI)], swallowing [(M.D. Anderson Dysphagia Inventory (MDADI)] and quality of life [European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Head and Neck Module (EORTC-H&N35)]. Outcome measures were completed pre-operatively and at 1-, 6-, and 12-months post-operatively. RESULTS: One hundred and seventeen patients undergoing partial glossectomy with reconstruction participated in this study. Results indicated no significant differences in swallowing function (MDADI and EORTC-H&N35 subscales) between baseline and 6 months post-surgery and no significant differences in speech function (SHI subscales) between baseline and 1 year post-surgery. Most quality of life domains (EORTC-H&N35 subscales) returned to baseline levels by 1 year post-operation, while difficulties with dry mouth and sticky saliva persisted. A clear time trend of adjuvant (chemo)radiation therapy negatively affecting dry mouth scores over time was identified in this study, while negative independent effects of chemoradiation on MDADI swallowing, and EORTC-H&N35 swallowing, eating, and opening mouth subscales were found. CONCLUSIONS: Assessment time influenced patient-reported speech, swallowing, and quality of life outcomes, while treatment (by time) effects were found for only swallowing and quality of life outcomes. Results of the present study will help guide clinical care and will be useful for patient counseling on expected short and long-term functional and quality of life outcomes of surgical and adjuvant treatment for oral cavity cancer.

Ratiometric Sensing and Imaging of Intracellular pH Using Polyethylenimine-Coated Photon Upconversion Nanoprobes.

Measurement of changes of pH at various intracellular compartments has potential to solve questions concerning the processing of endocytosed material, regulation of the acidification process, and also acidification of vesicles destined for exocytosis. To monitor these events, the nanosized optical pH probes need to provide ratiometric signals in the optically transparent biological window, target to all relevant intracellular compartments, and to facilitate imaging at subcellular resolution without interference from the biological matrix. To meet these criteria we sensitize the surface conjugated pH sensitive indicator via an upconversion process utilizing an energy transfer from the nanoparticle to the indicator. Live cells were imaged with a scanning confocal microscope equipped with a low-energy 980 nm laser excitation, which facilitated high resolution and penetration depth into the specimen, and low phototoxicity needed for long-term imaging. Our upconversion nanoparticle resonance energy transfer based sensor with polyethylenimine-coating provides high colloidal stability, enhanced cellular uptake, and distribution across cellular compartments. This distribution was modulated with membrane integrity perturbing treatment that resulted into total loss of lysosomal compartments and a dramatic pH shift of endosomal compartments. These nanoprobes are well suited for detection of pH changes in in vitro models with high biological background fluorescence and in in vivo applications, e.g., for the bioimaging of small animal models.

Spectrally and Spatially Multiplexed Serological Array-in-Well Assay Utilizing Two-Color Upconversion Luminescence Imaging.

We demonstrate a simple dual-mode multiplexed array-in-well immunoassay for simultaneous classification and detection of serum IgG and IgM antibodies against influenza A and human adenoviruses based on the color and position of the upconversion luminescence on the array. Biotinylated influenza A/H1N1 and A/H5N1 as well as adenovirus serotype 2 and 5 hexon antigens along with positive and negative controls were printed in an array format onto the bottom of streptavidin-coated microtiter wells. The anti-influenza A and antiadenovirus antibodies present in the sample were captured to the array and detected with antihuman antibody-coated upconverting nanophosphors (UCNPs). The green emitting UCNPs (NaYF4:Yb(3+),Er(3+)) coated with antihuman IgG and blue emitting UCNPs (NaYF4:Yb(3+),Tm(3+)) coated with antihuman IgM were used to detect human IgG and IgM antibodies, respectively. The emission of the bound UCNPs was imaged free of autofluorescence with anti-Stokes photoluminescence microwell imager. No spectral cross-talk was observed between green and blue emitting UCNPs. Also the cross-reactivities between UCNP-conjugates and immobilized human IgG and IgM antibodies were negligible. Position of the signal on the array defined the antigen specificity and the antibody class was defined by the color of the upconversion luminescence. This technology could be used for differentiation between acute infection from past infection and immunity. Additionally, the class of the antibody response can be used for the differentiation between primary and secondary infections, hence, facilitating epidemiological seroprevalence studies.

Effects of blood sample anticoagulants on lateral flow assays using luminescent photon-upconverting and Eu(III) nanoparticle reporters.

Many quantitative and semiquantitative lateral flow (LF) assays have been introduced for clinical analytes such as biomarkers for cancer or acute myocardial infarction (AMI). Various detection technologies involving quantitative analyzing devices have been reported to have sufficient analytical sensitivity and quantification capability for clinical point-of-care tests. Fluorescence-based detection technologies such as quantum dots, Eu(III) nanoparticles, and photon-upconverting nanoparticles (UCNPs) have been introduced as promising solutions for point-of-care devices because of their high detectability by optical sensors. Lateral flow assays can be used for various sample types, e.g., urine, saliva, cerebrospinal fluid, and blood. This study focuses on the properties of serum and plasma because of their relevance in cancer and AMI diagnostics. The limit of detection was compared in LF assays having Eu(III) nanoparticles or UCNPs as reporters and the antibody configurations for two different analytes (prostate-specific antigen and cardiac troponin I (cTnI)). The results indicate a significant effect of anticoagulants in venipuncture tubes. The samples in K3EDTA tubes resulted in significant interference by decreased reporter particle mobility, and thus the limit of detection was up to eightfold less sensitive compared to serum samples. Despite the matrix interference in the cTnI assay with UCNP reporters, limits of detection of 41 ng/L with serum and 66 ng/L with the Li-heparin sample were obtained.

Array-in-well serodiagnostic assay utilizing upconverting phosphor label technology.

In this study, a multiplex serological array-in-well assay was constructed for simultaneous detection of serum IgG antibodies against parvovirus B19 and human adenovirus. The array was prepared in streptavidin-coated 96-well microtiter plates by spotting biotinylated parvovirus B19 virus-like-particles, adenovirus type 2 and 5 hexon antigens, negative control of human serum albumin and positive controls of human IgG and anti-human IgG antibodies on the bottom of each well in an array format with a printable area of 2 mm × 2 mm. The array-in-well assay was evaluated with serum samples (n=89) of different antibody status as determined by commercial enzyme immunoassay for parvovirus IgG, and by in-house enzyme immunoassay for adenovirus IgG. The bound serum anti-parvovirus IgG, anti-adenovirus IgG, and total IgG antibodies were detected with anti-human IgG antibody coated photon upconverting nanoparticles and the assay was measured with an anti-Stokes photoluminescence imager. Detection of specific antibodies by the multiplex array-in-well assay was in good agreement (100% for parvovirus B19 and 96% for adenovirus) with the reference results. In conclusion, the array-in-well with upconverting phosphor reporter technology was able to detect antiviral antibodies in human sera, and represents an efficient serodiagnostic concept that is a promising new tool for multiplex serology.

An integrated closed-tube 2-plex PCR amplification and hybridization assay with switchable lanthanide luminescence based spatial detection.

Switchable lanthanide luminescence is a binary probe technology that inherently enables a high signal modulation in separation-free detection of DNA targets. A luminescent lanthanide complex is formed only when the two probes hybridize adjacently to their target DNA. We have now further adapted this technology for the first time in the integration of a 2-plex polymerase chain reaction (PCR) amplification and hybridization-based solid-phase detection of the amplification products of the Staphylococcus aureus gyrB gene and an internal amplification control (IAC). The assay was performed in a sealed polypropylene PCR chip containing a flat-bottom reaction chamber with two immobilized capture probe spots. The surface of the reaction chamber was functionalized with NHS-PEG-azide and alkyne-modified capture probes for each amplicon, labeled with a light harvesting antenna ligand, and covalently attached as spots to the azide-modified reaction chamber using a copper(i)-catalyzed azide-alkyne cycloaddition. Asymmetric duplex-PCR was then performed with no template, one template or both templates present and with a europium ion carrier chelate labeled probe for each amplicon in the reaction. After amplification europium fluorescence was measured by scanning the reaction chamber as a 10 × 10 raster with 0.6 mm resolution in time-resolved mode. With this assay we were able to co-amplify and detect the amplification products of the gyrB target from 100, 1000 and 10,000 copies of isolated S. aureus DNA together with the amplification products from the initial 5000 copies of the synthetic IAC template in the same sealed reaction chamber. The addition of 10,000 copies of isolated non-target Escherichia coli DNA in the same reaction with 5000 copies of the synthetic IAC template did not interfere with the amplification or detection of the IAC. The dynamic range of the assay for the synthetic S. aureus gyrB target was three orders of magnitude and the limit of detection of 8 pM was obtained. This proof-of-concept study shows that the switchable lanthanide luminescent probes enable separation-free array-based multiplexed detection of the amplification products in a closed-tube PCR which can enable a higher degree of multiplexing than is currently feasible by using different spectrally separated fluorescent probes.