Clinical Features of Paediatric Inflammatory Epidermolysis Bullosa Acquisita: A Case Series Study.

Epidermolysis bullosa acquisita (EBA) rarely develops in childhood. This study retrospectively recruited paediatric patients with EBA (age ≤ 16 years), diagnosed by clinical and histopathological features and results of immunofluorescence, immunoblotting and enzyme-linked immunosorbent assay (ELISA), and reviews their clinical manifestations, histopathology, immunological features, and responses to various treatments. All 7 included patients presented with inflammatory EBA. Among them, 3 had a bullous pemphigoid-like phenotype. Pathologically, in addition to dermal-epidermal blistering, in all patients, the distribution of neutrophils was superficial perivascular or interstitial, or in the dermal papilla. Mixed neutrophils and eosinophils were detected in 2 of the 3 patients with bullous pemphigoid-like phenotypes. In addition to treatment with glucocorticoids, dapsone was administered in 4 patients, while thalidomide and sulfasalazine were administered in 1 patient. All patients responded to the these therapies. Relapse was mainly due to reduction and cessation of glucocorticoids. In conclusion, EBA in childhood may be unique, and thus distinct from its adult counterpart. Specific treatment and follow-up protocols are required for therapy of this rare autoimmune skin disease in children.

Bacterial Skin Infections in Hospitalized Patients with Bullous Pemphigoid.

OBJECTIVE: To explore the features and risk factors of bacterial skin infections (BSIs) in hospitalized patients with bullous pemphigoid (BP). METHODS: Records were retrospectively reviewed for 110 hospitalized patients with BP admitted to Peking University First Hospital between 2013 and 2019. Bacterial species and drug resistance were assessed, and then the underlying risk factors for BSIs were evaluated. RESULTS: Infections were present in 40% (44/110) of the patients. Staphylococcus aureus (72.7%, 32/44) was the most common bacterium, and it was highly resistant to penicillin (81.3%, 26/32), erythromycin (62.5%, 20/32), and clindamycin (56.3%, 18/32), but 100.0% sensitive to vancomycin and tigecycline. Coronary heart disease (P = .02; odds ratio [OR], 12.68), multisystem comorbidities (P = .02; OR, 3.67), hypoalbuminemia (P = .04; OR, 3.70), high levels of anti-BP180 antibodies (>112.4 U/mL; P = .003; OR, 6.43), and season (spring: reference; summer: P = .002; OR, 23.58; autumn: P = .02; OR, 12.19; winter: P = .02; OR, 13.19) were significantly associated with BSIs. CONCLUSIONS: Hospitalized patients with BP had a high incidence of BSIs, and those patients with underlying risk factors require careful management to prevent and control BSIs.

Features and associated factors of bacterial skin infections in hospitalized patients with pemphigus: a single-center retrospective study.

BACKGROUND: Infections were the primary cause of death (34.3-55.5%) in patients with pemphigus. Skin was usually the origin of infections. The study aimed to explore features and associated factors of bacterial skin infections (BSIs) in inpatients with pemphigus. METHODS: One hundred and seventy-seven inpatients with pemphigus hospitalizing from November 2014 to April 2019 were continuously recruited through Peking University First Hospital's inpatient records inpatients with pemphigus hospitalizing from November 2014 to April 2019 were continuously recruited through Peking University First Hospital's inpatient records. Then, we retrieved the clinical and laboratory data to explore the characteristics and associated factors of BSIs. RESULTS: Of patients enrolled, pemphigus vulgaris (PV, n = 142) and pemphigus foliaceus (PF, n = 9) were most common, followed by pemphigus erythematosus (PE, n = 25) and pemphigus vegetans (Pveg, n = 1). Eighty-seven of 177 (49.2%) inpatients developed BSIs, and they had a longer length of stay compared with inpatients without BSIs (median: 18.9 vs. 14.1 days, p = 0.008). Staphylococcus aureus was the most common bacteria (71.3%, 62/87) and highly resistant to penicillin (91.9%, 57/62). Higher levels of anti-Dsg1 autoantibodies (> 124.2 U/mL) (p < 0.001, odds ratio [OR] = 3.564, 95% confidence interval [CI]: 1.784-7.123) and anti-Dsg3 autoantibodies (> 169.5 U/mL) (p = 0.03, OR = 2.074, 95% CI: 1.084-3.969) were underlying risk factors of BSIs when analyzed by binary regression analysis. As for Gram's stain of bacteria, females had a lower rate of Gram-positive infections (p = 0.03). Patients using oral antibiotics (p = 0.05) had a higher rate of Gram-negative infections. Inpatients who were hospitalized in other hospitals within 2 weeks before the current admission had a higher rate of Gram-negative and co-infections (p = 0.03). CONCLUSIONS: Inpatients with pemphigus had a high incidence of BSIs. Some factors were associated with the susceptibility of BSIs and bacterial species.

Features and Risk Factors for Paraneoplastic Autoimmune Multiorgan Syndrome in 145 Chinese Patients.

Paraneoplastic autoimmune multiorgan syndrome is a complex and deadly disease. We retrospectively reviewed the clinical features and risk factors for paraneoplastic autoimmune multiorgan syndrome in 145 Chinese patients. The most common neoplasm was Castleman disease (56%), and patients with Castleman disease tended to be younger (≤ 42 years old: 83% vs. 29%) and to have a greater proportions of lichen planus-like lesions (47% vs. 27%) and bronchiolitis obliterans (49% vs. 29%), compared to other neoplasm-associated patients. Among all 145 patients in the study, the survival rates were 84% at 1 year, 65% at 3 years, and 54% at 5 years. Kaplan-Meier curve analysis revealed that mortality was associated with older age (> 42 years), neoplasm type, labial lesions, and larger skin lesion area (> 17.5% of the body surface area). However, only older age and larger skin lesion area were independent factors associated with mortality in multivariate analysis. We suggest that patients with Castleman disease and paraneoplastic autoimmune multiorgan syndrome have many unique characteristics and the underlying risk factors for death require further exploration.

Microfluidic electrophoretic non-enzymatic kanamycin assay making use of a stirring bar functionalized with gold-labeled aptamer, of a fluorescent DNA probe, and of signal amplification via hybridization chain reaction.

The authors describe an enzyme-free aptamer-based assay for the determination of the model antibiotic kanamycin (Kana). The method is making use of (a) microfluidic chip electrophoresis; (b) a stirring bar carrying a gold-labeled aptamer probe, and (c) the hybridization chain reaction (HCR) for signal amplification. Firstly, a stirring bar (length: 1 cm; diameter: 0.2 mm) was modified with a large amount of duplex DNA and then hybridized with aptamer and its partially complementary chains (cDNA). In the presence of Kana, the binding between the Kana and aptamer unwinds the duplex structures and releases a corresponding amount of cDNA into the supernatant. The released cDNA triggers the HCR in the presence of H1 and H2 DNA hairpin to produce a large amount of duplex DNA chains with different lengths. At the same time, the amounts of H1 and H2 are reduced. The decreased signal of H1/H2 after several HCR cycles can be used to quantify kana in the 1 pg·mL-1 to 10 ng·mL-1, with a detection limit of 0.29 pg·mL-1. The signal is generated by reading the fluorescence, best at excitation/emission maxima of 470/525 nm. The whole detection process takes 3 min only. The assay was employed to the detection of Kana in spiked milk and fish samples. Results are consistent with those of an enzyme linked immunosorbent assay. The assay has high throughput, high selectivity, and high amplification capability. Graphical abstract Schematic of a stirring bar functionalized with gold-labeled aptamer acting as the capture probe. It can capture the target and release primer simultaneously. The primer triggers the hybridization chain reaction inducing the consumption of H1 and H2. After a certain reaction time, the mixture is injected into the MCE platform for microfluidic electrophoretic separation and fluorometric detection.

Serum Human Beta-Defensin-2 Is a Possible Biomarker for Monitoring Response to JAK Inhibitor in Psoriasis Patients.

AIMS: To analyse the correlation between serum human beta-defensin-2 (hBD-2) levels and response to JAK inhibitor in psoriasis. METHODS: We evaluated the psoriasis area and severity index (PASI) and serum hBD-2 levels of 18 psoriasis patients randomized to receive placebo or tofacitinib 5 or 10 mg b.i.d. at baseline, week 8, and week 16. Serum hBD-2 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The PASI achieved a dramatic reduction after tofacitinib 5 or 10 mg b.i.d. treatment for 16 weeks (p < 0.05). Serum hBD-2 levels significantly decreased in patients treated with tofacitinib 10 mg b.i.d. compared with baseline and the placebo-treated patients (p < 0.05). A significant correlation was found between hBD-2 levels and PASI (r = 0.52, p < 0.01). A serum hBD-2 level of 1,255.45 pg/mL was a cut-off between mild and moderate-to-severe psoriasis in ROC analysis. CONCLUSIONS: Serum hBD-2 level might be a possible biomarker for monitoring psoriasis treatment response and differentiating mild from moderate-to-severe psoriasis.

[Primary cutaneous diffuse large B-cell lymphoma, leg type: a study of clinicopathology, immunophenotype and gene rearrangement].

OBJECTIVE: To study the clinicopathologic features, immunophenotype and gene rearrangement of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL). METHODS: Seven cases of PCLBCL were enrolled into the study. Clinicopathologic analysis, immunohistochemical staining and gene rearrangement for IgH and Igκ were undertaken in the study. RESULTS: All the seven cases were male, and the median age was 72 years. Patients usually presented with multiple purple tumors, nodules, papules and infiltrative plaques. Two patients had a history of leg injury before onset, and one had mosquito bites. Histologically, the tumor involved the dermis and subcutis with dense and diffuse infiltrative pattern composing of centroblasts and/or immunoblasts. Immunohistochemical staining showed that seven cases (7/7) expressed CD20, six (6/6) expressed bcl-2, four (4/4) expressed MUM-1, four (4/5) expressed CD79a, four (4/5) expressed PAX-5 and four (4/6) expressed bcl-6, respectively. All cases did not express CD3ε, CD45RO, CD10 and CD30. IgH gene rearranged bands were detected in three (3/6) cases and Igκ was detected in one (1/5) case. Six of the seven cases died and the remaining patient, who was 44-year-old, was alive after 22 months of follow-up. CONCLUSIONS: PCLBCL is rare, predominantly affects elderly male patients. PCLBCL has poor prognosis and high mortality, but younger patients seem to have better prognosis. Some cases had a history of trauma or mosquito bites. The relationship between the history and the onset of PCLBCL needs further evaluation.

Whole-Exome Sequencing Reveals the Genomic Profile and IL6ST Variants as a Prognostic Biomarker of Paraneoplastic Pemphigus-Associated Unicentric Castleman Disease.

Unicentric Castleman disease (UCD) is a rare lymphoproliferative disorder. Paraneoplastic pemphigus (PNP) is a major complication associated with poor UCD prognosis. However, the genomic profiles and prognostic biomarkers of PNP-associated UCD remain unclear. In this study, we performed whole-exome sequencing analysis for 28 matched tumor-normal pairs and 9 tumor-only samples to define the genomic landscape of Chinese patients with PNP-associated UCD. An integrative analysis was performed to identify somatic variants, the mutational signatures, and key pathways in tumors. Besides, we analyzed the relationship among mutated genes, clinical characteristics, and prognosis. Sixty-one somatic mutant genes were identified in >1 patient with PNP-associated UCD. Specifically, IL6ST and PDGFRB were the most frequently mutated genes (32%), followed by DPP6 (18%) and MUC4 (18%). Signaling molecules and interactions, cellular processes, and signal transduction pathways were enriched. Furthermore, we found that poor overall survival was related to IL6ST variants (P = .02). Finally, we classified PNP-associated UCD into 4 genomic subgroups: IL6ST, PDGFRB, IL6ST-PDGFRB, and an unknown subgroup. In summary, we defined the molecular profile of PNP-associated UCD and identified a potential molecular biomarker for predicting prognosis, which may provide therapeutic targets for treating this severe disorder.

Evaluation of Dupilumab in Patients With Bullous Pemphigoid.

Importance: Dupilumab is a theoretically novel therapy for bullous pemphigoid (BP). However, its effectiveness and safety have yet to be confirmed in a large-scale study. Objective: To assess the efficacy and safety of dupilumab in patients with BP and evaluate factors that potentially affect short-term and long-term outcomes. Design, Setting, and Participants: A retrospective cohort study was conducted from January 1, 2021, to July 31, 2022. The median (IQR) follow-up period was 24.6 (11.5-38.4) weeks. This multicenter study was performed in 6 dermatology departments of the National Autoimmune Bullous Diseases Cooperative Group of China. Adult patients with BP that received 300 mg of dupilumab every 2 weeks following an initial dose of 600 mg were included. Patients were eligible if they had a clinical presentation of BP combined with immunological or pathological evidence. Patients with drug-induced BP, with less than 4 weeks of follow-up, and who received dupilumab or any other biologics within 6 months were excluded. Main Outcomes and Measures: The primary outcome was the proportion of patients who achieved disease control within 4 weeks. Disease control was defined as the absence of new lesions and pruritus, combined with the healing of existing lesions. Complete remission rates, relapse rates, changes in Bullous Pemphigoid Disease Area Index (BPDAI) scores, itching numerical rating scale (NRS) scores, laboratory results within 64 weeks, and adverse events (AEs) were also assessed. Results: Among 146 patients (median [IQR] age, 73 [64-85] years; 86 [58.9%] male patients) included in the study, 127 (87.0%) patients achieved disease control within 4 weeks, with a median (IQR) time of 14 (7-14) days. A total of 52 (35.6%) patients achieved complete remission, and 13 (8.9%) patients relapsed during the observation period. The complete remission rate and cumulative relapse rate at week 64 were 62.5% (5 of 8) and 30.9%, respectively. There was rapid and sustained improvement in clinical indicators and laboratory examination results after dupilumab treatment, including BPDAI scores, itching NRS scores, serum anti-BP180 and anti-BP230 antibodies, total IgE levels, and eosinophil count. Of these 146 patients, 107 (73.3%) did not report any AEs. The most common AEs were infections and eosinophilia. Serum anti-BP180 antibody levels of greater than 50 relative units (RU)/mL (OR, 3.63; 95% CI, 0.97-12.61; P = .045) were associated with 4-week disease control, and male patients were more likely to relapse (HR, 10.97; 95% CI, 1.42-84.92; P = .02). Conclusions and Relevance: In this retrospective cohort study, dupilumab treatment was associated with improved clinical symptoms in patients with BP. The safety profile was favorable, although concurrent infection and eosinophilia might pose potential concerns. This study suggests that patients with anti-BP180 antibody levels of at least 50 RU/mL and female sex may respond better.

Evaluation of clinical factors and outcome of systemic glucocorticoid therapy discontinuation in patients with pemphigus achieving complete remission.

Therapy discontinuation of systemic glucocorticoid treatment for pemphigus remains uncertain at the clinical end point of complete remission. The objective of this study was to identify the factors associated with achieving complete remission off therapy (CROT) and analyze the occurrence of relapse after therapy discontinuation. A retrospective cohort study was conducted at the Department of Dermatology of Peking University First Hospital. A total of 447 patients with pemphigus treated from 2005 to 2020 were identified. Univariate and multivariate analyses were conducted to analyze the associated factors of CROT and to evaluate the outcomes. The mean age was 48 years (±13.4 years), and 54.6% of the patients were women. During a median follow-up of 59 months (43-87.5 months), 160 of 447 (35.8%) patients achieved CROT after a median treatment duration of 51 months (38-66.2 months). Patients with a shorter therapy duration to complete remission on minimal therapy and negative desmoglein antibodies tested in remission were more likely to achieve early CROT. Thirty-five of 160 (21.9%) patients experienced relapse after CROT. Patients who discontinued therapy without guidance experienced significantly faster and higher occurrences of relapse than those withdrawing under guidance (log-rank p = 0.01). Minimal therapy maintenance ≤8 months from complete remission on minimal therapy and positive desmoglein antibodies tested at withdrawal increased the risk of early relapse after CROT.

Evaluation of the immune colloidal gold technique for BP180-NC16A-specific antibodies in the quick diagnosis and monitoring of bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) mostly involves elderly patients. The diagnosis of BP requires special immunological tests, which makes some patients unable to be diagnosed and treated timely. OBJECTIVE: The accuracy and application value of immune colloidal gold technique (ICGT) in BP were evaluated. The colloidal gold was conjugated with recombinant BP180 NC16A protein and mouse IgG antibody. As the test and control lines, the mouse-anti-human IgG and goat-anti-mouse IgG, respectively, were blotted on the nitrocellulose membrane. METHODS: 414 serum samples of consecutive patients with suspected BP and 15 samples from healthy donors were recruited. The consistency between ICGT and ELISA, and between serum and plasma/whole blood were evaluated. Subgroup analyses were performed in terms of clinical characteristics. We also followed up 65 BP patients' strip results to explore the predictive value of ICGT. RESULTS: Strong agreements between ICGT and ELISA(κ = 0.902) and between plasma/whole blood and serum samples (κ = 0.980) with good stability were observed. The ICGT achieved sensitivity of 93.9%, and specificity of 97.6%. In subgroup analysis, the sensitivity was significantly higher in older patients (96.3%), and with more typical lesions such as blisters (96.2%) and erosions (92.4%). In follow-up, we also found BP patients who kept ICGT-negative in remission state all got consecutive positive strips 1-3 weeks prior to mild new activity or flare. CONCLUSION: ICGT shows high potential as a rapid and stable option for the diagnosis and monitoring of BP. Further investigations are needed to re-evaluate this technique in a prospective study with a multicenter design.

Paraneoplastic Pemphigus Autoantibodies Against C-terminus of Desmoplakin Induced Acantholysis In Vitro and In Vivo.

Paraneoplastic pemphigus (PNP) is an autoimmune bullous disease associated with underlying neoplasms and characterized by antibodies against desmoglein 3 (Dsg 3) and plakins. Autoantibodies against desmoglein 3 in sera of patients with PNP have been proven to cause acantholysis in vivo in neonatal mice. As a member of the plakin family, autoantibodies against desmoplakin were detected frequently by immunoprecipitation in the sera of PNP. The recombinant C-terminus of desmoplakin was expressed and purified to adsorb the specific autoantibodies against the C-terminus of desmoplakin. In vitro dispase-dependent keratinocyte dissociation assay and in vivo IgG passive transfer into neonatal mice assay were performed, followed by the electronic microscopy examination and TUNEL assay. We found that anti-C terminus of desmoplakin autoantibodies caused blisters and acantholysis in mice skin at a dose-dependent manner. Moreover, dissociated fragments were observed after incubation with the purified IgG against desmoplakin, compared with normal human IgG (P-value =0.0207). The electronic microscopy examination showed the disconnection of keratin intermediate filaments from desmosomes. Lastly, apoptosis of keratinocytes in the TUNEL assay was all detected in the skins of neonatal mice after injection of the anti-C terminus of desmoplakin autoantibodies. Taken together, the study suggests that autoantibodies against the C-terminus of desmoplakin might be pathogenic in PNP.

[Detection of serum desmoglein antibody level using enzyme-linked immunosorbent assay (ELISA) for monitoring disease activity in patients with pemphigus vulgaris].

OBJECTIVE: Pemphigus is an autoimmune blistering disease of skin and mucous membranes. Pemphigus vulgaris (PV) is a major subtype of pemphigus, which is histologically characterized by suprabasal acantholysis. The major antigen in PV is desmosomal glycoproteins desmoglein (Dsg) 3. The autoantibodies against Dsg3 cause loss of adhesion between keratinocytes. Some PV patients also have circulating anti-Dsg1 autoantibodies. Enzyme-linked immunosorbent assays (ELISAs) with recombinant Dsg3 and Dsg1 are highly sensitive and specific for detecting anti-Dsg3 and anti-Dsg1 autoantibodies in PV patients. To evaluate the role of desmosomal glycoproteins desmoglein (Dsg3) ELISA and Dsg1 ELISA for detecting anti-Dsg3 and anti-Dsg1 autoantibodies in monitoring disease activity in Pemphigus vulgaris patients. METHODS: Twenty PV patients with long-term follow-up were included. We tested their serial sera with modified Dsg3 ELISA (MESACUP Desmoglein TEST "Dsg3", Medical & Biological Laboratories Co. LTD.), Dsg1 ELISA(MESACUP Desmoglein TEST "Dsg1", Medical & Biological Laboratories Co. LTD.) and indirect immunofluorescence (IIF). Then we analyzed the correlation between Dsg3 ELISA index values, Dsg1 ELISA index values, IIF titres and disease activity scores (ABSIS) along the time course. RESULTS: There were significant correlations between Dsg3 ELISA index values, Dsg1 ELISA index values, IIF titres and disease activity scores (both skin scores and oral scores) (P<0.01) along the time course. Significant differences of Dsg3 ELISA index values, Dsg1 ELISA index values and IIF titres between active time-point group and clinical remission time-point group were also observed (P<0.01). We found that Dsg3 ELISA index values, Dsg1 ELISA index values and IIF titres fluctuated in parallel with disease activity, and ELISA index values were superior to IIF titres. CONCLUSION: Dsg3 ELISA index values fluctuating in parallel with disease activity are useful to monitor disease activity, predict flares or relapses and plan the schedules for tapering the drugs.

BIOCHIP mosaic for the diagnosis of autoimmune bullous diseases in Chinese patients.

BACKGROUND: Autoimmune blistering diseases (AIBDs) are a group of fatal diseases with specific autoantibodies. BIOCHIP mosaic is a novel and all-in-one measure used for the rapid diagnosis of AIBDs. OBJECTIVES: To evaluate the diagnostic accuracy based on BIOCHIP mosaic (FA1501-1005-60) in Chinese patients with AIBDs. MATERIALS AND METHODS: Seventy-seven patients with AIBDs and 20 controls were enrolled. The BIOCHIP mosaic was performed using both serum and plasma samples. RESULTS: Based on BIOCHIP mosaic, the data from paired plasma and serum samples demonstrated a high degree of concordance (Cohen's kappa = 0.896-1.000) for autoantibodies against Dsg1, Dsg3, BP180-NC16A-4X, BP230gC, prickle-cell desmosomes, and pemphigoid antigens. Moreover, BIOCHIP mosaic also demonstrated a high degree of consistency for the detection rate of anti-Dsg1, Dsg3, plakins, BP180-NC16A-4X and non-collagenous domain of type VII collagen autoantibodies for the diagnosis of pemphigus foliaceus (77.3%), pemphigus vulgaris (88.6%), paraneoplastic pemphigus (100.0%), bullous pemphigoid (92.8%) and epidermolysis bullosa acquisita (99.0%), respectively. CONCLUSION: Using BIOCHIP mosaic, serum and plasma samples may be used interchangeably at 1/10 dilution. Overall, the BIOCHIP mosaic was shown to be a useful and accurate tool for the diagnosis of AIBDs.

Predominant Stroma-Rich Feature in Hyaline Vascular Variant of Castleman Disease Is Associated With Paraneoplastic Pemphigus.

OBJECTIVES: We aimed to describe the clinical and histopathologic features of Castleman disease (CD), particularly emphasizing its associations with paraneoplastic pemphigus (PNP) and prognosis. METHODS: We retrospectively enrolled 123 CD patients at our center. Clinical, pathologic, and laboratory data were reviewed. RESULTS: Fifty percent of the patients had PNP. Compared with those without PNP, patients with PNP-associated CD had more hyaline vascular (HV) variants (83.9% vs 57.4%), fewer mixed cellular variants (16.1% vs 24.6%), and no plasmacytic variants (0% vs 18.0%). Thirty-eight of 87 patients with the HV variant of CD (HV-CD) had stroma-rich (SR) features, and the incidence rate was higher in those with PNP-associated CD than in those without PNP (48.4% vs 13.1%, P < .001). The SR variant was associated with higher PNP-associated IgG titers than SR absence before surgery (median 1:160 vs 1:80, P = .019) or after surgery (median 1:160 vs 1:40, P = .013). The SR variant was also an unfavorable prognostic factor for CD survival in univariate analysis. The 3-year survival rates were 47.5% among those with PNP and 87.7% among those without PNP (P < .001). CONCLUSIONS: PNP is associated with specific subtypes of CD and affects survival. The SR variant of HV-CD positively correlates with the incidence of PNP.

Microfluidic Chip for Multiplex Detection of Trace Chemical Contaminants Based on Magnetic Encoded Aptamer Probes and Multibranched DNA Nanostructures as Signal Tags.

The development of multiplex assays to simultaneously monitor multiclass chemical contaminants that commonly coexist in foods, such as heavy metal ions, antibiotics, and estrogen residues, is gaining attention. Here, a microfluidic chip (MC)-based multianalysis method coupled with magnetic encoded aptamer probes was used for simultaneous detection of kanamycin, 17ß-estradiol, and lead ion (Pb2+). Using this innovative strategy, the magnetic bead (MB)-based encoded probes labeled with aptamer hybrid chains were first used to selectively capture multiple targets, followed by generating single-stranded primers. The primers triggered a multibranched hybridization chain reaction (mHCR). Finally, three kinds of complementary strands (C-DNAs) with different lengths were hybridized with the arms of the mHCR products to form three types of multibranched DNA nanostructures. The decrement signals of C-DNAs were employed for qualification of targets. As the signal tags corresponded to different targets, the DNA nanostructures realized "one target for the decrease of massive C-DNAs" to improve sensitivity. The use of MB-based encoded probes could achieve magnetic separation to eliminate interference in the complex. The detection limits of this method were 1.76 × 10-4 nM (kanamycin), 1.18 × 10-4 nM (17ß-estradiol), and 1.29 × 10-4 nM (lead ion). Furthermore, the MC platform is reusable and can be used for more than 4000 samples. The assay combining the MC with MB-based encoded probes with multibranched DNA signal tags offers a universal, reusable, and high-throughput detection platform for screening multiclass chemical contaminants in food samples with complex matrices.

Microchip electrophoresis based multiplexed assay for silver and mercury ions simultaneous detection in complex samples using a stirring bar modified with encoded hairpin probes for specific extraction.

It is crucially important to rapidly, simultaneously, and sensitively determine trace amounts of heavy metal ions in complex samples. Herein, a stirring bar modified with two kinds of encoded hairpin DNA probes (H0 and H0') was used in a multiplexed strategy allowing for specific extraction of Hg2+ and Ag+ coupled to microchip electrophoresis (MCE) separation and LED induced fluorescence (LIF) detection. The extraction step utilizes stir bars, which are functionalized with designed hairpin DNA probes (H0 with TT and H0' with CC mismatches in stems). This allows the specific capture of Hg2+ and Ag+ through CAg+C and THg2+T interactions. These complexes are then enzymatically degraded by the action of exonuclease III (Exo III). The ions released during this enzymatic reaction can initiate a new cycle of interactions with hairpin structures and enzymatic reactions and so on. This cyclic step is specific to the presence of Hg2+ and Ag+ and represents the first round of amplification of the presence of the selected ions. The resulting single strand DNAs on the stirring bars after enzymatic degradation were used in the second step as primers to trigger the catalytic hairpin assembly (CHA) in the presence of a couple of hairpin structures in solution. Such a reaction allows producing duplexes that can be monitored by MCE-LIF. The fluorescence intensity of CHA products (IP) increased and that of hairpin DNAs (IR) decreased with the increase of target concentrations. The signal ratios (IP/IR and IP'/IR') consisted of targets. The assay was employed for Hg2+ and Ag+ detection in several mediums including water, milk, and fish samples with complex matrices. The results showed that the assay could avoid matrix interference to increase the sensitivity. Therefore, the multiplexed assay was ideal to simultaneously and quickly detect metal ions in complex samples.