Infectivity of Coronavirus Disease 2019: A Prospective Cohort Study in the Korean Metropolitan Area.

BACKGROUND: Although several characteristics of coronavirus disease 2019 (COVID-19), an ongoing pandemic disease, have been identified, data on the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited. METHODS: This prospective cohort study was conducted to analyze the infectivity of SARS-CoV-2 based on data of all patients diagnosed with COVID-19 confirmed using real-time polymerase chain reaction test from January to April 2020 in Gyeonggi-do, the largest province in Korea. RESULTS: Of the 502 patients, 298 consisting of 106 clusters with 5,909 contacts were included. Of these, 277 (93.0%) were symptomatic, and the most common symptoms were cough, fever, sputum, sore throat, and headache. A total of 94 patients (31.5%) had pneumonia, while 8 (2.7%) died during the follow-up period. The secondary attack rate (SAR) in the study population was 3.5% (204/5,909). In exposure settings, the SAR was higher in religious gathering (13.5% [95% confidence interval, 10.7-16.8%]), workplaces (8.49% [95% CI, 6.08-11.74%]), and schools (6.38% [95% CI, 3.39-11.69%]) than in health care facilities (1.92% [95% CI, 1.45-2.55%]). Sore throat at any period, dyspnea at diagnosis or any period, lower cycle threshold value in the lower respiratory tract samples, leukocytosis, and higher bilirubin levels were associated with higher infectivity of COVID-19. The presence of symptoms was not related to the infectivity. CONCLUSION: In establishing the infection control strategies for COVID-19, the variables associated with high infectivity may be considered.

Clinical Course and Outcomes of 3,060 Patients with Coronavirus Disease 2019 in Korea, January-May 2020.

BACKGROUND: The fatality rate of patients with coronavirus disease 2019 (COVID-19) varies among countries owing to demographics, patient comorbidities, surge capacity of healthcare systems, and the quality of medical care. We assessed the clinical outcomes of patients with COVID-19 during the first wave of the epidemic in Korea. METHODS: Using a modified World Health Organization clinical record form, we obtained clinical data for 3,060 patients with COVID-19 treated at 55 hospitals in Korea. Disease severity scores were defined as: 1) no limitation of daily activities; 2) limitation of daily activities but no need for supplemental oxygen; 3) supplemental oxygen via nasal cannula; 4) supplemental oxygen via facial mask; 5) non-invasive mechanical ventilation; 6) invasive mechanical ventilation; 7) multi-organ failure or extracorporeal membrane oxygenation therapy; and 8) death. Recovery was defined as a severity score of 1 or 2, or discharge and release from isolation. RESULTS: The median age of the patients was 43 years of age; 43.6% were male. The median time from illness onset to admission was 5 days. Of the patients with a disease severity score of 3-4 on admission, 65 (71.5%) of the 91 patients recovered, and 7 (7.7%) died due to illness by day 28. Of the patients with disease severity scores of 5-7, 7 (19.5%) of the 36 patients recovered, and 8 (22.2%) died due to illness by day 28. None of the 1,324 patients who were < 50 years of age died; in contrast, the fatality rate due to illness by day 28 was 0.5% (2/375), 0.9% (2/215), 5.8% (6/104), and 14.0% (7/50) for the patients aged 50-59, 60-69, 70-79, and ≥ 80 years of age, respectively. CONCLUSION: In Korea, almost all patients of < 50 years of age with COVID-19 recovered without supplemental oxygen. In patients of ≥ 50 years of age, the fatality rate increased with age, reaching 14% in patients of ≥ 80 years of age.

Larger medial femoral to tibial condylar dimension may trigger posterior root tear of medial meniscus.

PURPOSE: The major meniscal functions are load bearing, load distribution, and shock absorption by increasing the tibiofemoral joint (TFJ) contact area and dissipating axial loads by conversion into hoop stresses. The increased hoop strain stretches the meniscus in outward direction towards radius, causing extrusion, which is associated with the root tear and resultant degenerative osteoarthritis. Since the larger contact area of medial TFJ may increase the hoop stresses, we hypothesized that the larger medial femoral to tibial condylar dimension would contribute to the development of medial meniscus posterior root tear (MMPRT). Thus, the purpose of the study was to assess the relationship between MMPRT and medial femoral to tibial condylar dimension. METHODS: A case-control study was conducted to compare medial femoral to tibial condylar dimensions of patients with complete MMPRT (n = 59) with those of demography-matched controls (n = 59) during the period from 2010 to 2013. In each patient, MRIs were reviewed and several parameters were measured including articulation width of medial femoral condyle (MFC) at 0°, 30°, 60°, and 90°, medial tibial condyle (MTC) width, degree of meniscal extrusion, and medial femoral to tibial condylar width ratio (MFC/MTC) at 0°, 30°, 60°, and 90°, respectively. Demographic and radiographic data were assessed. RESULTS: A larger medial femoral to tibial condylar dimension was associated with MMPRT at 0° and 30° knee angles. Patients with MFC/MTC greater than 0.9 at 0° also showed about 2.5-fold increase in the chance of MMPRT. Those with meniscal extrusion greater than 3 mm also had about 17.1 times greater chance for the presence of MMPRT accordingly. CONCLUSIONS: A larger medial femoral to tibial condylar dimension may be considered as one of the regional contributors to the outbreak of MMPRT, and medial femoral to tibial condylar width ratio greater than 0.9 at 0° knee angle may be considered as a significant risk factor for MMPRT. LEVEL OF EVIDENCE: III.

Aurantio-obtusin stimulates chemotactic migration and differentiation of MC3T3-E1 osteoblast cells.

Osteoporosis is one of the major metabolic bone diseases and is among the most challenging noncommunicable diseases to treat. Although there is an increasing interest in identifying bioactive molecules for the prevention and management of osteoporosis, such studies principally focus only on differentiation and mineralization of osteoblasts or inhibition of osteoclast activity. Stimulation of osteoblast migration must be a promising osteoanabolic strategy for improved metabolic bone disease therapy. In this study, we show that an anthraquinone derivative, aurantio-obtusin, stimulated chemotactic migration of MC3T3-E1 osteoblast cells in a concentration-dependent manner. The use of a real-time chemotaxis analyzing system, TAXIScan, facilitated the evaluation of both velocity and directionality of osteoblast migration in response to the compound. Besides migration, the compound stimulated osteoblast differentiation and mineralization. Taken together, the data presented in this paper demonstrate that aurantio-obtusin is a promising osteoanabolic compound of natural origin with potential therapeutic applications in the prevention of osteoporosis and other metabolic bone diseases.

Difference in knee rotation between total and unicompartmental knee arthroplasties during stair climbing.

PURPOSE: The purpose of this study was to compare knee kinematics during stair walking in patients with simultaneous total knee arthroplasty (TKA) and unicompartmental knee arthroplasties (UKA). It was hypothesized that UKA would reproduce more normalized knee kinematics than TKA during stair ascent and descent. METHODS: Six patients who received UKA in one knee and TKA in the other knee were included in the study. For this study, a four-step staircase was assembled with two force platforms being positioned at the centre of the second and third steps. Each patient was attached with 16 reflective markers at both lower extremities and was asked to perform five roundtrip trials of stair climbing. Kinematic parameters including stance duration, knee angle, vertical ground reaction force (GRF), joint reaction force, and moments were obtained and analysed using a10-camera motion system (VICON, Oxford, UK). Nonparametric Friedman test was used to compare the results between two arthroplasty methods and between stair ascent and descent. RESULTS: Compared to TKA, UKA knees exhibited significantly greater degree of rotation in transverse planes (5.0 degrees during ascent and 6.0 degrees during descent on average), but showed no difference in terms of the other parameters. When comparing the results during stair ascent with descent, overall greater knee angle, vertical GRF, joint reaction force, and moment were observed during stair descent. CONCLUSIONS: Both UKA and TKA knees have shown overall similar knee kinematics, though UKA knee may allow greater degree of rotation freedom, which resembles normal knee kinematics during stair walking.

Prognostic value of central blood pressure on the outcomes of embolic stroke of undetermined source.

We investigated the prognostic impact of central blood pressure (BP) on outcomes in patients with embolic stroke of undetermined source (ESUS). The prognostic value of central BP according to ESUS subtype was also evaluated. We recruited patients with ESUS and data on their central BP parameters (central systolic BP [SBP], central diastolic BP [DBP], central pulse pressure [PP], augmentation pressure [AP], and augmentation index [AIx]) during admission. ESUS subtype classification was arteriogenic embolism, minor cardioembolism, two or more causes, and no cause. Major adverse cardiovascular event (MACE) was defined as recurrent stroke, acute coronary syndrome, hospitalization for heart failure, or death. Over a median of 45.8 months, 746 patients with ESUS were enrolled and followed up. Patients had a mean age of 62.8 years, and 62.2% were male. Multivariable Cox regression analysis showed that central SBP and PP were associated with MACE. All-cause mortality was independently associated with AIx. In patients with no cause ESUS, central SBP and PP, AP, and AIx were independently associated with MACE. AP and AIx were independently associated with all-cause mortality (all p < 0.05). We demonstrated that central BP can predict poor long-term prognosis in patients with ESUS, especially those with the no cause ESUS subtype.

Solid phase DNA extraction with a flexible bead-packed microfluidic device to detect methicillin-resistant Staphylococcus aureus in nasal swabs.

We have developed a bead-packed microfluidic device with a built-in flexible wall to automate extraction of nucleic acids from methicillin-resistant Staphylococcus aureus (MRSA) in nasal swabs. The flexible polydimethylsiloxane (PDMS) membrane was designed to manipulate the surface-to-volume ratio (SVR) of bead-packed chambers in the range of 0.05 to 0.15 (µm(-1)) for a typical solid phase extraction protocol composed of binding, washing, and eluting. In particular, the pneumatically assisted close packing of beads led to an invariant SVR (0.15 µm(-1)) even with different bead amounts (10-16 mg), which allowed for consistent operation of the device and improved capture efficiency for bacteria cells. Furthermore, vigorous mixing by asynchronous membrane vibration enabled ca. 90% DNA recovery with ca. 10 µL of liquid solution from the captured cells on the bead surfaces. The full processes to detect MRSA in nasal swabs, i.e., nasal swab collection, prefiltration, on-chip DNA extraction, and real-time polymerase chain reaction (PCR) amplification, were successfully constructed and carried out to validate the capability to detect MRSA in nasal swab samples. This flexible microdevice provided an excellent analytical PCR detection sensitivity of ca. 61 CFU/swab with 95% confidence interval, which turned out to be higher than or similar to that of the commercial DNA-based MRSA detection techniques. This excellent performance would be attributed to the capability of the flexible bead-packed microdevice to enrich the analyte from a large initial sample (e.g., 1 mL) into a microscale volume of eluate (e.g., 10 µL). The proposed microdevice will find many applications as a solid phase extraction method toward various sample-to-answer systems.

Association between Low Ankle-Brachial Index and Poor Outcomes in Patients with Embolic Stroke of Undetermined Source.

We investigated the association of low ankle-brachial index (ABI < 0.9) with major adverse cardiovascular events (MACE) and all-cause mortality in patients with embolic stroke of undetermined source (ESUS) as well as whether the association differed by ESUS subtype. This retrospective single-center study included ESUS patients who underwent transesophageal echocardiography and ABI during hospitalization. ESUS was classified as ESUS with minor cardioembolic source, arteriogenic embolism, two or more causes, or no cause. Arteriogenic embolism was defined and classified as complex aortic or non-stenotic relevant artery plaque. MACE was defined as stroke recurrence, acute coronary syndrome, hospitalization for heart failure, or death. Overall, 829 patients were included, with a median follow-up of 45.8 months. Of these, 42 (5.1%) and 370 (44.6%) had low ABI and arteriogenic embolism, respectively. ABI < 0.9 was independently associated with MACE (hazard ratio [HR]: 2.038, 95% confidence interval [CI]: 1.093−3.801) and all-cause mortality (HR: 3.608, 95% CI: 1.538−8.465) according to the multivariable Cox regression analysis. Between ESUS subtypes, low ABI was independently associated with MACE (HR: 2.513, 95% CI: 1.257−5.023) and all-cause mortality (HR: 5.681, 95% CI: 2.151−15.008) in arteriogenic embolism patients, especially in those with complex aortic plaque. However, in non-arteriogenic embolism patients, low ABI was not related to MACE and mortality. In ESUS patients, low ABI was linked to MACE and all-cause mortality, especially in those with arteriogenic embolisms from complex aortic plaque.

Clinical findings, viral load, and outcomes of COVID-19: Comparison of patients with negative and positive initial chest computed tomography.

Reports detailing the clinical characteristics, viral load, and outcomes of patients with normal initial chest CT findings are lacking. We sought to compare the differences in clinical findings, viral loads, and outcomes between patients with confirmed COVID-19 who initially tested negative on chest CT (CT negative) with patients who tested initially positive on chest CT (CT positive). The clinical data, viral loads, and outcomes of initial CT-positive and CT-negative patients examined between January 2020 and April 2020 were retrospectively compared. The efficacy of viral load (cyclic threshold value [Ct value]) in predicting pneumonia was evaluated using receiver operating characteristic (ROC) curve and area under the curve (AUC). In total, 128 patients underwent initial chest CT (mean age, 54.3 ± 19.0 years, 50% male). Of those, 36 were initially CT negative, and 92 were CT positive. The CT-positive patients were significantly older (P < .001) than the CT-negative patients. Only age was significantly associated with the initial presence of pneumonia (odds ratio, 1.060; confidence interval (CI), 1.020-1-102; P = .003). In addition, age (OR, 1.062; CI, 1.014-1.112; P = .011), fever at diagnosis (OR, 6.689; CI, 1.715-26.096; P = .006), and CRP level (OR, 1.393; CI, 1.150-1.687; P = .001) were significantly associated with the need for O2 therapy. Viral load was significantly higher in the CT-positive group than in the CT-negative group (P = .017). The cutoff Ct value for predicting the presence of pneumonia was 27.71. Outcomes including the mean hospital stay, intensive care unit admission, and O2 therapy were significantly worse in the CT-positive group than in the CT-negative group (all P < .05). In conclusion, initially CT-negative patients showed better outcomes than initially CT-positive patients. Age was significantly associated with the initial presence of pneumonia, and viral load may help in predicting the initial presence of pneumonia.

Mass-Synthesized Solution-Processable Polyimide Gate Dielectrics for Electrically Stable Operating OFETs and Integrated Circuits.

Polyimides (PIs) are widely utilized polymeric materials for high-temperature plastics, adhesives, dielectrics, nonlinear optical materials, flexible hard-coating films, and substrates for flexible electronics. PIs can be facilely mass-produced through factory methods, so the industrial application value is limitless. Herein, we synthesized a typical poly(amic acid) (PAA) precursor-based solution through an industrialized reactor for mass production and applied the prepared solution to form thin films of PI using thermal imidization. The deposited PI thin films were successfully applied as gate dielectrics for organic field-effect transistors (OFETs). The PI layers showed suitable characteristics for dielectrics, such as a smooth surface, low leakage current density, uniform dielectric constant (k) values regardless of frequency, and compatibility with organic semiconductors. Utilizing this PI layer, we were able to fabricate electrically stable operated OFETs, which exhibited a threshold voltage shift lower than 1 V under bias-stress conditions and a field-effect mobility of 4.29 cm2 V-1 s-1. Moreover, integrated logic gates were manufactured using these well-operated OFETs and displayed suitable operation behavior.

Clinical and Laboratory Factors Associated with Symptom Development in Asymptomatic COVID-19 Patients at the Time of Diagnosis.

In preparation for the surge of coronavirus disease 2019 (COVID-19), it is crucial to allocate medical resources efficiently for distinguishing people who remain asymptomatic until the end of the disease. Between January 27, 2020, and April 21, 2020, 517 COVID-19 cases from 13 healthcare facilities in Gyeonggi province, Korea, were identified out of which the epidemiologic and clinical information of 66 asymptomatic patients at the time of diagnosis were analyzed retrospectively. An exposure-diagnosis interval within 7 days and abnormal aspartate aminotransferase levels were identified as characteristic symptom development in asymptomatic COVID-19 patients. If asymptomatic patients without these characteristics at the time of diagnosis could be differentiated early, more medical resources could be secured for mild or moderate cases in this COVID-19 surge.

Electrochemical cell lysis device for DNA extraction.

We present a novel electrochemical cell lysis device to prepare DNA samples for lab-on-a-chip (LOC) applications. It utilizes the electrolysis of saline solution to generate hydroxide ions (OH(-)) at the cathode as alkaline lytic agents. Cathode and anode chambers are separated by a negatively-charged ion exchangeable polymer diaphragm to maintain the high pH level for efficient cell lysis in the cathode chamber, to prevent inflow of PCR-amplification inhibitors from the anode chamber, and to minimize binding of DNA molecules. Electric current flow and pH maintenance, which depended on the device design, were two important parameters of the device performance. After optimizing the design and visually confirming cell lysis of Chinese hamster ovary (CHO) cells in a very short amount of time, we directly electrolyzed four bacterial cell types suspended in saline solution. Real-time PCR (qPCR) analysis showed that our device could lyse both gram-positive and gram-negative bacterial cells with higher efficiency than other common methods and could detect DNA on the microlitre scale. Our data demonstrate several advantages of the proposed device: absence of cell lysis chemicals and heating; no adverse effects on PCR amplification; low DNA loss; low voltage and power consumption; and rapid processing. The device could potentially be applied as an on-chip DNA extraction component.

Toward a disposable low-cost LOC device: heterogeneous polymer micro valve and pump fabricated by UV/ozone-assisted thermal fusion bonding.

Herein, a heterogeneous polymer micro valve and pump with a polypropylene (PP) membrane was developed in a low-cost manner via UV/ozone-assisted thermal fusion bonding. The proposed fabrication technique allowed for a geometrically selective bonding; consequently, the membrane was prevented from bonding with the valve seat of the diaphragm micro-valve, without patterning a protection layer or introducing an additional structure. The developed device withstands 480 kPa of static pressure and up to 350 kPa of a vibration pressure, providing sufficient bonding strength for microfluidic actuations. The fabricated micro valve and pump are fully characterized and compared with a poly(dimethylsiloxane) (PDMS) membrane glass device, showing comparable valving and pumping performance. As a result, the robust PP membrane micro valve and pump are simply implemented in a facile manner, and demonstrated excellent performance, which is highly desirable for mass production of disposable lab-on-a-chip (LOC) devices.

Bacterial DNA sample preparation from whole blood using surface-modified Si pillar arrays.

A novel bacterial DNA sample preparation device for molecular diagnostics has been developed. On the basis of optimized conditions for bacterial adhesion, surface-modified silicon pillar arrays for bacterial cell capture were fabricated, and their ability to capture bacterial cells was demonstrated. The capture efficiency for bacterial cells such as Escherichia coli, Staphylococcus epidermidis, and Streptococcus mutans in buffer solution was over 75% with a flow rate of 400 microL/min. Moreover, the proposed method captured E. coli cells present in 50% whole blood effectively. The captured cells from whole blood were then in- situ lyzed on the surface of the microchip, and the eluted DNA was successfully amplified by qPCR. These results demonstrate that the full process of pathogen capture to DNA isolation from whole blood could be automated in a single microchip.

Isolation of total RNA from Escherichia coli using kosmotropic Hofmeister salts.

Most of the widely used RNA isolation methods involve the use of toxic chemicals, including chaotropic salts and phenol. In an effort to solve this problem, we studied an alternative method to purify total RNA without any harmful chemicals. This method was based on silica spin columns and kosmotropic Hofmeister salts. The RNA yield was comparable to that of the commercially available RNeasy Mini Kit (Qiagen) at salt concentrations between 0.5 and 1.0 M. Furthermore, the current method allowed the isolation of small RNA molecules together with all RNA molecules longer than 200 nt.

Reversible capture of genomic DNA by a Nafion-coated electrode.

A method in which an electrode itself is used as the sample preparation microchip is described. The gold electrode was coated with an ion-permeable polymer, Nafion, to prevent the permanent adsorption and destruction of DNA. The modified electrode was able to capture as much DNA as the bare gold electrode and to release the captured DNA effectively, whereas the bare gold electrode could not release bound DNA. The elution efficiency was greater than 70% for the Nafion-coated electrode, whereas it was less than 10% for the bare electrode. The DNA obtained was undamaged and could be amplified by polymerase chain reaction.

Otologic manifestations of Hunter syndrome and their relationship with speech development.

The excessive storage of mucopolysaccharide in Hunter syndrome leads to various otologic manifestations. We interviewed 19 patients with Hunter syndrome to assess their otologic problems, and conducted audiologic tests and temporal bone CT. Patients with the intermediate or severe form exhibited severe speech delay by more than 2 years (12/14 patients). However, in patients with the mild form (5/5), speech development was not much disturbed (2/5), although otoscopic findings were similar. The hearing threshold determined by the auditory brainstem response differed significantly between the mild and intermediate/severe forms (p < 0.05). Therefore, patients with the mild form may benefit from active otologic intervention such as VT insertion, amplification, and speech therapy.

Di(2-ethylhexyl)phthalate leached from medical PVC devices serves as a substrate and inhibitor for the P-glycoprotein.

A di(2-ethylhexyl)phthalate (DEHP) was accidentally extracted from plastics in the process of purification of chemosensitizers reversing P-glycoprotein (Pgp)-mediated multidrug resistance (MDR). The purpose of this study was to investigate the Pgp-reversal activities of phthalates, which are endocrine-disrupting chemicals, by utilizing the Pgp-overexpressing leukemic cell line AML-2/D100. The phthalates includes DEHP, diethyl phthalate (DEP) and dibutyl phthalate (DBP). Of the tested phthalates, DEHP showed the highest Pgp-reversal activity and DEP the most potent drug-accumulating activity. On the other hand, they did not show any chemosensitizing activity against multidrug resistance associated protein-mediated MDR. The complete inhibition of Pgp by verapamil increased the cytotoxicity of DEHP, but neither DEP nor DBP had this effect, suggesting that DEHP alone may be a possible substrate for the Pgp. DEHP showed higher hydrophobicity than the other phthalates when determined by reverse phase-HPLC. In addition, DEHP, but not the others increased the ATPase activity in a concentration-dependent manner. This is the first report that phthalates can reverse Pgp-mediated MDR by increasing drug accumulation, as well as serving as substrates for the Pgp. It is thought that the hydrophobic characteristics of phthalates could play an important role in Pgp-inhibitory activity. Therefore, pharmaco- and toxicokinetic interactions between phthalates leached from medical PVC devices and substrates for the Pgp should be kept in mind.

A Longitudinal Study on the Effects of Negative Rearing Experiences on Adolescents' Social Withdrawal and Aggression.

BACKGROUND: Children who have experienced negative rearing behaviors show a lack of self-confidence due to emotional instability and are reserved in interpersonal relationships. This can lead to failure in social adaptation and a high risk of depression, suicide, criminal acts, and anti-social behaviors. Therefore, this study aims to analyze the effects of experiencing negative parental rearing behaviors, such as neglect and abuse, on adolescents' social withdrawal and aggression, by utilizing multivariate latent growth models. METHODS: Data from the Korean Children and Youth Panel Study (KCYPS), a survey conducted by the National Youth Policy Institute targeting a cohort of three different age groups (grade 1, grade 4, and grade 7), from 2010 to 2016 was used. Multi-stage stratified sampling methods were used in the KCYPS, which surveyed the students and parents of the selected grade levels. This study analyzed the data for grade 7, from second year (grade 8) to fourth year (grade 10). RESULTS: Negative rearing experiences had a significant effect on social withdrawal and aggression, and this influence was shown to persist over the long term. CONCLUSION: This study examined the long-term developmental trajectory in the relationship between risk factors for adolescent development. Furthermore, the relationship between risk factors was shown to have not only short term but long-term effects as well, which reinforces the limitations of previous studies.

Poly(dimethyl siloxane)-based protein chip for simultaneous detection of multiple samples: use of glycidyl methacrylate photopolymer for site-specific protein immobilization.

This paper describes fabrication of a poly(dimethyl siloxane) (PDMS)-based chip to analyze multiple protein interactions utilizing glycidyl methacrylate (GMA) photopolymer for a site-specific immobilization of capture proteins in a closed system. First, using one direction channels of a PDMS mold having cross-channels, GMA micropads were prepared by photopolymerizing GMA solution by 365 nm light irradiation at predetermined positions. After the first mold was replaced with a second mold having higher height or directly without mold changing, capture proteins were allowed to be covalently immobilized onto the surface of the epoxide-activated GMA pads. Following immobilization, poly(ethylene glycol) diacrylate (PEG-DA) precursor was photopolymerized at specific regions to generate plugs for prevention of mixing between different sample injection channels, diminishing the need of a mold changing for sample injections. Final chip was assembled by connecting separated sample injection channels using a connector mold. The viability of this strategy was successfully demonstrated by simultaneous detection of two different antigen-antibody interactions.