RESUMO
BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune bullous disease mediated by autoantibodies against desmoglein 3 (DSG3). Inducible costimulator (ICOS) is a costimulatory receptor expressed on T cells and influences the activity of T follicular helper (TFH) cells in various autoimmune diseases, but the roles of ICOS and TFH cells in PV remain unclear. OBJECTIVE: We examined the immunological characteristics, antigen specificity, and pathogenicity of CD4+ T-cell subpopulations, as well as the therapeutic effect of anti-ICOS blocking antibodies in PV. METHODS: A mouse model of PV was established by adoptive transfer of immune cells from the skin-draining lymph nodes or spleens of DSG3-expressing skin-grafted Dsg3-/- mice into Rag1-/- mice. The TFH cells and CD4+ T cells in PBMCs from PV patients were examined by flow cytometry. RESULTS: Among CD4+ T cells from the mouse model, ICOS-positive TFH cells were associated with B-cell differentiation and were required for disease induction. Using an MHC class II tetramer, DSG3-specific ICOS+ TFH cells were found to be associated with anti-DSG3 antibody production and expanded in the absence of B cells. In human PV, the frequency of ICOS+CXCR5+PD-1+ memory CD4+ T cells correlated with the autoantibody level. Treatment with anti-ICOS blocking antibodies targeting ICOS+ TFH cells decreased the anti-DSG3 antibody level and delayed disease progression in vivo. CONCLUSIONS: Mouse Dsg3-specific ICOS+ TFH cells and human ICOS+CXCR5+PD-1+ TH cells are associated with the anti-DSG3 antibody response in PV. ICOS expressed on CXCR5+PD-1+ TH cells may be a therapeutic target for PV.
Assuntos
Anti-Inflamatórios/uso terapêutico , Anticorpos Bloqueadores/uso terapêutico , Desmogleína 3/metabolismo , Centro Germinativo/imunologia , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Pênfigo/terapia , Células Th1/metabolismo , Animais , Autoanticorpos/metabolismo , Desmogleína 3/genética , Modelos Animais de Doenças , Progressão da Doença , Citometria de Fluxo , Humanos , Memória Imunológica , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Camundongos , Camundongos Knockout , Pênfigo/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores CXCR5/metabolismo , Células Th1/imunologiaRESUMO
The number of pesticides used in agriculture is increasing steadily, leading to contamination of soil and drinking water. Herein, we present a microfluidic platform to detect the extent of contamination in soil samples. A microchip capillary electrophoresis system with in-channel electrodes was fabricated for label-free electroanalytical detection of triazine herbicides. The sample mixture contained three representative triazines: simazine, atrazine and ametryn. The electropherogram for each individual injection of simazine, atrazine and ametryn showed peaks at 58, 66 and 72 s whereas a mixture of them showed distinct peaks at 59, 67 and 71 s respectively. The technique as such may prove to be a useful qualitative and quantitative tool for the similar environmental pollutants.
Assuntos
Eletroforese Capilar/métodos , Monitoramento Ambiental/métodos , Herbicidas/análise , Poluentes do Solo/análise , Triazinas/análise , Agricultura , Atrazina/análise , Atrazina/química , Herbicidas/química , Simazina/análise , Simazina/química , Poluentes do Solo/química , Triazinas/químicaRESUMO
PURPOSE: Clinical implications of neoadjuvant immunotherapy in patients with locally advanced but resectable head and neck squamous cell carcinoma (HNSCC) remain largely unexplored. PATIENTS AND METHODS: Patients with resectable HNSCC were randomized to receive a single dose of preoperative durvalumab (D) with or without tremelimumab (T) before resection, followed by postoperative (chemo)radiotherapy based on multidisciplinary discretion and 1-year D treatment. Artificial intelligence (AI)-powered spatial distribution analysis of tumor-infiltrating lymphocytes and high-dimensional profiling of circulating immune cells tracked dynamic intratumoral and systemic immune responses. RESULTS: Of the 48 patients enrolled (D, 24 patients; D+T, 24 patients), 45 underwent surgical resection per protocol (D, 21 patients; D+T, 24 patients). D±T had a favorable safety profile and did not delay surgery. Distant recurrence-free survival (DRFS) was significantly better in patients treated with D+T than in those treated with D monotherapy. AI-powered whole-slide image analysis demonstrated that D+T significantly reshaped the tumor microenvironment toward immune-inflamed phenotypes, in contrast with the D monotherapy or cytotoxic chemotherapy. High-dimensional profiling of circulating immune cells revealed a significant expansion of T-cell subsets characterized by proliferation and activation in response to D+T therapy, which was rare following D monotherapy. Importantly, expansion of specific clusters in CD8+ T cells and non-regulatory CD4+ T cells with activation and exhaustion programs was associated with prolonged DRFS in patients treated with D+T. CONCLUSIONS: Preoperative D±T is feasible and may benefit patients with resectable HNSCC. Distinct changes in the tumor microenvironment and circulating immune cells were induced by each treatment regimen, warranting further investigation.
Assuntos
Anticorpos Monoclonais Humanizados , Anticorpos Monoclonais , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias de Cabeça e Pescoço , Terapia Neoadjuvante , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Masculino , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/terapia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Pessoa de Meia-Idade , Feminino , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/uso terapêutico , Idoso , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Neoplasias de Cabeça e Pescoço/terapia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Terapia Neoadjuvante/métodos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Adulto , Microambiente Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Bax inhibitor-1 (BI-1), a member of the BI-1 family of integral membrane proteins, was originally identified as an inhibitor of stress-induced cell death in mammalian cells. Previous studies have shown that the withdrawal of leukemia inhibitory factor (LIF) results in differentiation of the majority of mouse embryonic stem (mES) cells into various cell lineages, while some ES cells die within 3days. Thus, to investigate the function of BI-1 in ES cell survival and neuronal differentiation, we generated mES cell lines that overexpress BI-1 or a carboxy-terminal BI-1ΔC mutant. Overexpression of BI-1 in mES cells significantly increased cell viability and resistance to apoptosis induced by LIF withdrawal, while the control vector or BI-1ΔC-overexpressing mES cells had no effect. Moreover, overexpression of BI-1 produced significant inhibition of the p38 mitogen-activated protein kinases (MAPK) pathway in response to LIF withdrawal, while activity of the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) MAPK pathway was increased. Interestingly, we found that BI-1-overexpressing cells showed higher expression levels of neuroectodermal markers (Otx1, Lmx1b, En1, Pax2, Wnt1, Sox1, and Nestin) and greater neuronal differentiation efficiency than control or BI-1ΔC-overexpressing mES cells did. Considering these findings, our results indicated that BI-1-modulated MAPK activity plays a key role in protecting mES cells from LIF-withdrawal-induced apoptosis and in promoting their differentiation toward neuronal lineages.
Assuntos
Apoptose , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neurônios/citologia , Animais , Western Blotting , Proliferação de Células , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Técnicas Imunoenzimáticas , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Proteínas de Membrana/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Neurônios/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUNDPemphigus, a rare autoimmune bullous disease mediated by antidesmoglein autoantibodies, can be controlled with systemic medication like rituximab and high-dose systemic corticosteroids combined with immunosuppressants. However, some patients continue to experience chronically recurrent blisters in a specific area and require long-term maintenance systemic therapy.METHODSSkin with chronic blisters was obtained from patients with pemphigus. Immunologic properties of the skin were analyzed by immunofluorescence staining, bulk and single-cell RNA and TCR sequencing, and a highly multiplex imaging technique known as CO-Detection by indEXing (CODEX). Functional analyses were performed by flow cytometry and bulk RNA-Seq using peripheral blood from healthy donors. Intralesional corticosteroid was injected into patient skin, and changes in chronically recurrent blisters were observed.RESULTSWe demonstrated the presence of skin tertiary lymphoid structures (TLSs) with desmoglein-specific B cells in chronic blisters from patients with pemphigus. In the skin TLSs, CD4+ T cells predominantly produced CXCL13. These clonally expanded CXCL13+CD4+ T cells exhibited features of activated Th1-like cells and downregulated genes associated with T cell receptor-mediated signaling. Tregs are in direct contact with CXCL13+CD4+ memory T cells and increased CXCL13 production of CD4+ T cells through IL-2 consumption and TGF-ß stimulation. Finally, intralesional corticosteroid injection improved chronic blisters and reduced skin TLSs in patients with pemphigus.CONCLUSIONThrough this study we conclude that skin TLSs are associated with the persistence of chronically recurrent blisters in patients with pemphigus, and the microenvironmental network involving CXCL13+CD4+ T cells and Tregs within these structures plays an important role in CXCL13 production.TRIAL REGISTRATIONClinicalTrials.gov NCT04509570.FUNDINGThis work was supported by National Research Foundation of South Korea (NRF-2021R1C1C1007179) and Korea Drug Development Fund, which is funded by Ministry of Science and ICT; Ministry of Trade, Industry, and Energy; and Ministry of Health and Welfare (grant RS-2022-00165917).
Assuntos
Doenças Autoimunes , Pênfigo , Humanos , Corticosteroides , Autoanticorpos , Doenças Autoimunes/tratamento farmacológico , Vesícula/tratamento farmacológico , Linfócitos T CD4-Positivos , Quimiocina CXCL13 , Desmogleína 3 , Pênfigo/tratamento farmacológicoRESUMO
Rapid and reliable molecular analysis of DNA for disease diagnosis is highly sought-after. FET-based sensors fulfill the demands of future point-of-care devices due to its sensitive charge sensing and possibility of integration with electronic instruments. However, most of the FETs are unstable in aqueous conditions, less sensitive and requires conventional Ag/AgCl electrode for gating. In this work, we propose a solution-gated graphene FET (SG-FET) for real-time monitoring of microscale loop-mediated isothermal amplification of DNA. The SG-FET was fabricated effortlessly with graphene as an active layer, on-chip co-planar electrodes, and polydimethylsiloxane-based microfluidic reservoir. A linear response of about 0.23V/pH was seen when the buffers from pH 5-9 were analyzed on the SG-FET. To evaluate the performance of SG-FET, we monitored the amplification of Lambda phage gene as a proof-of-concept. During amplification, protons are released, which gradually alters the Dirac point voltage (VDirac) of SG-FET. The resulting device was highly sensitive with a femto-level limit of detection. The SG-FET could easily produce a positive signal within 16.5min of amplification. An amplification of 10ng/µl DNA for 1h produced a ∆VDirac of 0.27V. The sensor was tested within a range of 2×102 copies/µl (10 fg/µl) to 2×108 copies/µl (10ng/µl) of target DNA. Development of this sensing technology could significantly lower the time, cost, and complications of DNA detection.
Assuntos
Técnicas Biossensoriais , DNA Viral/isolamento & purificação , Grafite/química , Dispositivos Lab-On-A-Chip , DNA Viral/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Soluções/químicaRESUMO
Most of the reported field effect transistors (FETs) fall short of a general method to uniquely specify and detect a target analyte. For this reason, we propose a pentacene-based FET with a graphene oxide support system (GOSS), composed of functionalized graphene oxide (GO) ink. The GOSS with a specific moiety group to capture the biomaterial of interest was inkjet printed on the pentacene FET. It provided modular receptor sites on the surface of pentacene, without alteration of the device. To evaluate the performance of a GOSS-pentacene FET biosensor, we detected the artificial DNA and circulating tumor cells as a proof-of-concept. The mobility of the FET dramatically changed upon capturing the target biomolecule on the GOSS. The FET exhibited high selectivity with 0.1 pmoles of the target DNA and a few cancer cells per detection volume. This study suggests a valuable sensor for medical diagnosis that can be mass produced effortlessly at low-cost.
RESUMO
In this study, we developed a capacitive sensor for the one-step and label-free detection of protein kinase A (PKA) enzyme. Metal-insulator-semiconductor (MIS) and electrolyte-insulator-semiconductor (EIS) are a simple electronic transducer, which allows efficient detection of the target analyte. For this reason, we performed a comparative sensing of PKA on the MIS and EIS capacitive sensor. The PKA-specific aptamer was used for the one-step detection. For the immobilization of thiolated aptamer, the MIS sensor contained a thin gold layer, whereas the EIS sensor had a self-aligned monolayer of gold nanoparticles. The interaction of aptamer and PKA changed the charge and density of the sensor surface. The quantitative detection of PKA was performed by analyzing the capacitance-voltage curve after the aptamer-PKA interaction. The MIS and EIS sensor showed a detection limit of 5 U/mL and 1 U/mL, respectively, for the detection of PKA. This study suggests valuable sensing platforms for the rapid and sensitive biochemical diagnosis.
Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Proteínas Quinases/análise , Aptâmeros de Peptídeos/química , Aptâmeros de Peptídeos/metabolismo , Desenho de Equipamento , Ouro/química , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Nanopartículas Metálicas/química , Proteínas Quinases/metabolismo , Semicondutores , Sensibilidade e Especificidade , Propriedades de SuperfícieRESUMO
Adenosine is one of the nucleoside which plays an important role in signal transduction and neuromodulation. This work proposes a simple electrochemical assay, comprising two enzymes and rhodium complex based electron transfer mediator, for the detection of adenosine. Sequential reaction of adenosine deaminase and L-glutamic dehydrogenase and the supporting cycle between ß-NADH and mediator enable quantitative analysis of adenosine. Role of electron transfer mediator is the conveyance of proton from electrode to ß-NAD(+) for regeneration of ß-NADH. The electrochemical characteristics of electron transfer mediator were also studied. Real-time adenosine detection was carried out using this multiple enzyme based chronoamperometric assay. The analysis results show a low limit of detection (140 µM) and good correspondence between current signal and the adenosine concentration (R (2) = 0.997).
Assuntos
Adenosina/análise , Técnicas Biossensoriais/métodos , Enzimas Imobilizadas/metabolismo , Compostos Organometálicos/química , Ródio/química , Adenosina/química , Eletroquímica , Transporte de Elétrons , Enzimas Imobilizadas/química , NAD/químicaRESUMO
Efficient maintenance of the undifferentiated status of embryonic stem cells (ESCs) may be important for preparation of high-quality cell sources that can be successfully used for stem cell research and therapy. Here we tried to identify a compound that can enhance the quality of pluripotent stem cells. Treatment of ESCs and induced pluripotent stem cells (iPSCs) with 3,2'-dihydroxyflavone (3,2'-DHF) led to increases in cell growth, colony formation, and cell proliferation. Treatment with 3,2'-DHF resulted in high expression of pluripotency markers (OCT4, SOX2, and NANOG) and significant activation (STAT3 and AKT) or suppression (GSK3ß and ERK) of self-renewal-related kinases. 3,2'-DHF-treated high-quality pluripotent stem cells also showed enhanced differentiation potential. In particular, treatment of iPSCs with 3,2'-DHF led to elevated expression of ectodermal differentiation markers and improved differentiation into fully matured neurons. Next, we investigated the in vivo effect of 3,2'-DHF-pretreated iPSCs (3,2'-DHF iPSCs) in a peripheral nerve injury model and found that transplantation of 3,2'-DHF iPSCs resulted in more efficient axonal regeneration and functional recovery than in controls. Upon histopathological and gene expression analyses, we found that transplantation of 3,2'-DHF iPSCs stimulated expression of cytokines, such as TNF-α, in the early phase of injury and successfully reduced convalescence time of the injured peripheral nerve, showing an effective neuroprotective property. Taken together, our data suggest that 3,2'-DHF can be used for more efficient maintenance of pluripotent stem cells as well as for further applications in stem cell research and therapy.
Assuntos
Proliferação de Células/efeitos dos fármacos , Flavonoides/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Axônios/fisiologia , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Masculino , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Ratos , Ratos Sprague-Dawley , Regeneração , Neuropatia Ciática/terapia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
A high-throughput (HTS) and high-content screening (HCS) campaign of a commercial library identified 2,3-dihydroimidazo[1,2-a]benzimidazole analogues as a novel class of anti-parasitic agents. A series of synthetic derivatives were evaluated for their in vitro anti-leishmanial and anti-trypanosomal activities against Leishmania donovani and Trypanosoma cruzi, which have been known as the causative parasites for visceral leishmaniasis and Chagas disease, respectively. In the case of Leishmania, the compounds were tested in both intracellular amastigote and extracellular promastigote assays. Compounds 4 and 24 showed promising anti-leishmanial activity against intracellular L. donovani (3.05 and 5.29 µM, respectively) and anti-trypanosomal activity against T. cruzi (1.10 and 2.10 µM, respectively) without serious cytotoxicity toward THP-1 and U2OS cell lines.
Assuntos
Antiparasitários/síntese química , Antiparasitários/farmacologia , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Compostos Heterocíclicos com 3 Anéis/síntese química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Leishmania donovani/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Antiparasitários/química , Benzimidazóis/química , Células Cultivadas , Relação Dose-Resposta a Droga , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-AtividadeRESUMO
Nano-scale materials are noted for unique properties, distinct from those of their bulk material equivalents. In this study, we prepared spherical silver nanoparticles (AgNPs) with an average size of about 30 nm and tested their potency to induce neuronal differentiation of SH-SY5Y cells. Human neuroblastoma SH-SY5Y cells are considered an ideal in vitro model for studying neurogenesis, as they can be maintained in an undifferentiated state or be induced to differentiate into neuron-like phenotypes in vitro by several differentiation-inducing agents. Treatment of SH-SY5Y cells by biologically synthesized AgNPs led to cell morphological changes and significant increase in neurite length and enhanced the expression of neuronal differentiation markers such as Map-2, ß-tubulin III, synaptophysin, neurogenin-1, Gap-43, and Drd-2. Furthermore, we observed an increase in generation of intracellular reactive oxygen species (ROS), activation of several kinases such as ERK and AKT, and downregulation of expression of dual-specificity phosphatases (DUSPs) in AgNPs-exposed SH-SY5Y cells. Our results suggest that AgNPs modulate the intracellular signaling pathways, leading to neuronal differentiation, and could be applied as promising nanomaterials for stem cell research and therapy.
Assuntos
Nanopartículas Metálicas/química , Neurônios/citologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Prata/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Proteína GAP-43/genética , Proteína GAP-43/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/metabolismo , Monoéster Fosfórico Hidrolases , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Sinaptofisina/genética , Sinaptofisina/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Sulfur-containing amino acids, such as cysteine and homocysteine play crucial roles in biological systems for the diagnosis of medical states. In this regard, this paper deals with separation, aliquot and detection of amino thiols on a microchip capillary electrophoresis with electrochemical detection in an inverted double Y-shaped microchannel. Unlike the conventional capillary electrophoresis, the modified microchannel design helps in storing the separated thiols in different reservoirs for further analysis, if required; and also eliminates the need of electrodes regeneration. The device was fabricated using conventional photolithographic technique which consisted of gold microelectrodes on a soda lime glass wafer and microchannels in PDMS mold. Multiple detections were performed using in-house fabricated dual potentiostat. Based on amperometric detection, cysteine and homocysteine were analyzed in 105 s and 120 s, respectively after diverting in branched channels. Repeated experiments proved the good reproducibility of the device. The device produced a linear response for both cysteine and homocysteine in electrochemical analysis. To prove the practicality of device, we also analyzed cysteine and homocysteine in real blood samples without any pre-treatment. Upon calculation, the device showed a very low limit of detection of 0.05 µM. The modified microchip design shall find a broad range of analytical applications involving assays of thiols and other biological compounds.
Assuntos
Técnicas Biossensoriais/instrumentação , Condutometria/instrumentação , Eletroforese Capilar/instrumentação , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Compostos de Sulfidrila/análise , Ação Capilar , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A fully integrated microchip for performing cell lysis, polymerase chain reaction (PCR) and quantitative analysis of DNA amplicons in a single step is described herein. The chip was built on glass substrate using an indium-tin-oxide (ITO) microheater and PDMS engraved microchannels, which integrated an electrochemical cell lysis zone, a continuous flow PCR module and capillary electrophoresis amperometric detection (CE-AD) system. The total length of the microchannel was 4625 mm for performing 25 cycles of flow-through PCR and was laid on a handheld form factor of 96 × 96 mm(2) area. The key to the fabrication of such a device lies in the use of a single medium to carry out different kinds of biochemical reactions and hence, a reagentless electrochemical cell lysis protocol was integrated on the microchip which was capable of lysing most cell types, including difficult to lyse gram positive bacteria. The lysate contained genomic DNA from a sample which was proven to be suitable for PCR reactions. Genetic analysis was successfully performed on the microchip with purified lambda phage genomic DNA and various cell types, including non-tumorigenic MCF-10A and tumorigenic MCF-7 human cell lines, gram negative bacteria Escherichia coli O157:H7, and gram positive bacteria Bacillus subtilis, at an optimized flow rate of 5 µl min(-1). For the detection of amplicon DNA, a CE-AD system was used, with semisolid alkaline agarose within the capillary microchannel to minimize interference from cell debris and for efficient resolution of DNA fragments. High signal to noise ratio during amperometric detection and the use of online FFT filtering protocol enhanced the limit of detection of DNA amplicons. Therefore, with a combination of portability, cost-effectiveness and performance, the proposed integrated PCR microchip can be used for one step genetic analysis of most of the cell types and will enable more accessible healthcare.