Handheld Real-Time PCR Device

Lab on a Chip PAPER Ci e his: Lab Chip,2016,16,586 Recei ed 18 h No embe 2015, Accep ed 16 h Decembe 2015 DOI: 10.1039/c5lc01415h www. sc.o g/loc Handheld eal- ime PCR de ice† Ch is ian D. Ah be g, a Bojan Robe Ilic, b And eas Manz a and Pa el Neužil* acd He e we epo one o he smalles eal- ime polyme ase chain eac ion (PCR) sys ems o da e wi h an app oxima e size o 100 mm ×60 mm ×33 mm. The sys em is an au onomous uni equi ing an ex e nal 12 V powe supply. Fou simul aneous eac ions a e pe o med in he o m o i ual eac ion chambe s (VRCs) whe e a ≈200 nL sample is co e ed wi h mine al oil and placed on a glass co e slip. Fas , 40 cycle ampli ica ion o an amplicon om he H7N9 gene was used o demons a e he PCR pe o mance. The s anda d cu e slope was −3.02 ±0.16 cycles a h eshold pe decade (mean ±s anda d de ia ion) co e- sponding o an ampli ica ion e iciency o 0.91 ±0.05 pe cycle (mean ±s anda d de ia ion). The PCR de- ice was capable o de ec ing a single deoxy ibonucleic acid (DNA) copy. These esul s u he sugges ha ou handheld PCR de ice may ha e b oad, echnologically- ele an applica ions ex ending o apid de ec- ion o in ec ious diseases in small clinics. The in en ion o polyme ase chain eac ion (PCR) 32 yea s ago is conside ed o be one o he g ea es in en ions o he las cen u y. 1 O e he yea s, many a ian s o he o iginal sys em ha e been de eloped. One o he mos impo an ad ancemen s is he eal- ime PCR analysis sys em. 2 The ap- p oach enables eal- ime PCR ampli ica ion, moni o ing, and quan i ica ion o he numbe o deoxy ibonucleic acid (DNA) copies in he sample unde conside a ion. This me hod is commonly e e ed o as quan i a i e PCR (qPCR). 2 The main ad an age o eal- ime PCR is he elimina ion o any pos -p o- cessing, such as elec opho esis o hyb idiza ion o de ec he PCR p oduc . The PCR eac ion is pe o med by he mal cycling in he p esence o speci ic oligonucleo ides, he enzyme polyme ase, ee nucleic acids and bi alen sal s such as MgSO 4 o MgCl 2 . This cock ail is commonly e e ed o as he PCR mas e mix. The de ec ion o PCR p oduc ampli ica ion is conduc ed by moni o ing he luo escence ampli ude du ing he PCR. In he p esence o an in e cala ing dye, such as SYBR G een I, he luo escence ampli ude is p opo ional o he concen a- ion o he DNA amplicon, he p oduc o he PCR. In o de o e i y ampli ica ion speci ici y, upon PCR comple ion, em- ploymen o an in e cala ing dye enables pe o ming mel ing cu e analysis (MCA). Ano he imp o emen o he PCR is he addi ion o he e e se ansc ip ase enzyme o he PCR cock- ail, o ming e e se ansc ip ion PCR (RT-PCR). PCR has become he me hod o choice o he de ec ion o DNA and RT-PCR o de ec RNA. These wo eac ions ha e e olu ionized gene ics. Fu he mo e, PCR has many di e se applica ions in in ec ious disease diagnos ics o de ec ion o i uses o bac e ia, 3 in o ensic science, 4,5 pa e ni y es s, 6 secu i y applica ions 7 and my iad o he comme cial applica- ions. 8 Comme cial sys ems a e ypically a he la ge able- op ools used o high h oughpu mass sc eenings and a e imp ac ical o use in poin -o -ca e applica ions (POC), whe e he mos impo an sys em pa ame e s a e po abili y and powe consump ion. The ques o a minia u ized PCR e - sion sui able o POC diagnos ics was ini ia ed a Law ence Li e mo e Labo a o ies 9,10 mo e han wo decades ago. Ag awal e al. ha e de eloped a pocke -sized con en ional PCR sys em 11 ha equi es ex ensi e sample pos -p ocessing o iden i y he p esence o an amplicon. In con as , eal- ime PCR elimina es he need o sample p ocessing once PCR is comple ed. Real- ime PCR sys ems consis o a hea e , empe a u e senso , and luo escence exci a ion and de ec ion uni . Tem- pe a u e cycling is pe o med by hea ing and cooling o sam- ples. Wi hin he PCR p ocess, he cooling a e is one o he p ima y limi ing ac o s. Bulky comme cial sys ems ha e la ge hea capaci ies, hence hea emo al is challenging, and is ypi- cally accomplished by using a he moelec ic coole (TEC), commonly known as he Pel ie elemen . Since hese bulky sys ems consume a conside able amoun o powe , hey a e gene ally unsui able o ield es ing POC applica ions. 586 |Lab Chip,2016,16,586–592 This jou nal is © The Royal Socie y o Chemis y 2016 a KIST-Eu ope, Mic o luidics G oup, Campus E7.1, 66111 Saa b ücken, Ge many. E-mail: [email p o ec ed] b Na ional Ins i u e o S anda d and Technology (NIST), Cen e o Nanoscale Science and Technology, 100 Bu eau D i e, MS 6201, Gai he sbu g, MD 20899- 6201, USA c B no Uni e si y o Technology (BUT), Cen al Eu opean Ins i u e o Technology (CEITEC), Technická 3058/10, CZ-616 00 B no, Czech Republic d No hwes e n Poly echnical Uni e si y (NPU), School o Mechanical Enginee ing, Depa men o Mic osys em Enginee ing, 127 Wes Youyi Road, Xi'an Shaanxi, 710072, PR China †Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: 10.1039/ c5lc01415h Open Access A icle. Published on 16 Decembe 2015. Downloaded on 23/06/2016 07:07:56. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online View Jou nal | View Issue Lab Chip, 2016, 16,586–592 | 587This jou nal is © The Royal Socie y o Chemis y 2016 Small PCR ins umen s a e o en based on mic o luidic de ices, so-called “lab-on-a-chip”de ices. 12 These sys ems comp ise wo majo g oups, spa ial-domain and ime-domain PCRs. On he one hand, ime-domain PCRs ha e a single hea e wi h samples placed in di ec con ac . He e, empe a- u e cycling is ca ied ou by changing he hea e elemen empe a u e. On he opposi e end o he spec um, he spa ial-domain PCR has se e al hea e s, each held a a di e - en empe a u e. In his scena io, empe a u e cycling is accomplished by mo ing samples be ween hea e s. A ypical ep esen a i e o a spa ial-domain sys em is he con inuous PCR-on-a-chip. 13 Wi hin his sys em, he sample low in he mic o luidic chip is posi ioned o e he hea e s, each kep a a di e en empe a u e. The sample lows h ough ubes, he eby achie ing he mal cycling. He e, PCR du a ion is only limi ed by he low a e and he hea ans e be ween he sample and he side walls, o bo h hea ing and cooling. The wo majo d awbacks o a low- h ough PCR a e sys em complexi y and a high likelihood o sample- o-sample c oss-con amina ion. An al e na i e e sion was in oduced a ew yea s ago whe e he sample was in he o m o a i ual eac ion cham- be (VRC). 14–16 The VRC sel -assembly sys em consis s o a wa e d ople co e ed wi h mine al oil, p e en ing wa e e ap- o a ion om he sample. In his scena io, he wa e d ople con ained he PCR mas e mix wi h a p e-de e mined num- be o DNA copies. He e, he VRC wi h DNA was sepa a ed om he mic omachined silicon hea e s by a disposable, hyd ophobically-coa ed mic oscope co e slip. To elimina e sample- o-sample con amina ion, he glass co e slip was a disposable pa o he sys em, and he e o e each co e slip was a single use componen . The sample con ained magne ic pa icles which acili a ed sample mo ion be ween hea e s. 17 A pocke -size eal- ime PCR sys em capable o p ocessing a single sample was in oduced a ew yea s la e . 18 The sys em had an in eg a ed minia u ized op ical de ec ion uni , LCD display and con ol elec onics. One o he key ea u es was he implemen a ion o lock-in ampli ica ion o op ical signal p ocessing. 17 The lock-in ampli ica ion ea u e allowed o ambien sys em ope a ion wi hou ligh p o ec ion, he eby ende ing he sys em obus and use - iendly. One o he sys- em d awbacks was p ocessing a single sample a a ime and u he mo e, he de ice was bulky. A p ac ical sys em o conduc PCR o POC applica ions e- qui es simul aneous p ocessing o 4 o mo e samples. Diag- noses o clinical samples should be concu en ly conduc ed wi h posi i e and nega i e con ol samples, he eby elimina - ing alse nega i e o posi i e e en s. In ou wo k, we in oduce a new po able PCR sys em (Fig. 1) capable o concu en ly analyzing ou ≈200 nL ol- ume samples. The sys em speed is de e mined by he hea ing and cooling a es. The hea ing a e collec i ely a ises om he VRC he mal capaci ance (H) and he dissipa ed Joule hea . The a e o passi e cooling applied wi hin ou sys em is gi en by he he mal ime cons an (τ) o he sys em, which is gi en by H/G, whe e Gis he he mal conduc ance. Since he speci ic hea o wa e is excep ionally la ge, he he mal p op- e ies o ou sys em a e s ongly dependen on he sample wa e olume. Consequen ly, a smalle sample size esul s in a as e sys em. 19 Sys em samples consis o a nega i e con ol, also called no empla e con ol (NTC), a posi i e con ol, and wo sam- ples o in e es . The ou sample sys em a chi ec u e ep e- sen s he minimal numbe o samples equi ed o p ac ical applica ions. Ou sys em conduc s 40 PCR cycles in less han ≈35 min, while simul aneously p ocessing he esul s. Fu - he mo e, ou po able eal- ime PCR is capable o de ec ing a single DNA copy. The PCR pe o mance was e alua ed by de ec ing a com- plemen a y DNA om he a ian in luenza i us (H7N9) as well as wo human ansc ip s, hypoxan hine phospho ibosyl ans e ase (HPRT) and glyce aldehyde-3- phospha e dehyd ogenase (GAPDH). To he bes o ou knowledge, ou sys em pla o m ep esen s he smalles eal- ime PCR sys em. Ou PCR ins umen has wo key ea u es: 1. The ou samples a e in he VRC o m and a e placed on a disposable glass co e slip o e mic omachined silicon hea e s. Upon PCR comple ion, he single-use, disposable glass elemen is emo ed and a new glass co e slip is placed on op o he silicon hea e . 2. The luo escence exci a ion/de ec ion sys em is based on a lock-in ampli ie , he eby ende ing he sys em immune o ambien ligh . The PCR ins umen is equipped wi h a g aphical 84 ×48 pixel liquid c ys al display (LCD) wi h a di- agonal size o ≈38.1 mm o show he eac ion p og ess and Fig. 1 (A) CAD design d awing o he handheld PCR. The illus a ion shows a display wi h a compa men accommoda ing 4 samples in he VRC o m. (B) Fab ica ed and assembled comple e eal- ime PCR de- ice packaged wi hin a 3D p in ed casing. Lab on a Chip Pape Open Access A icle. Published on 16 Decembe 2015. Downloaded on 23/06/2016 07:07:56. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online 588 |Lab Chip,2016,16,586–592 This jou nal is © The Royal Socie y o Chemis y 2016 inal esul s. The cap u ed da a is s o ed in an in e nal mem- o y and can be uploaded o ex e nal p ocessing ia a uni e - sal se ial bus (USB). The sys em is powe ed by an ex e nal 12 V ba e y. Sys em se up Ou cu en sys em has wo new key ea u es: an in eg a ed op ical head and simpli ied con ol elec onics. 1. In eg a ed op ical head A luo escence de ec ion sys em o a single spo equi es a ligh sou ce, h ee il e s (exci a ion, dich oic mi o and emission) and a de ec o . We edesigned he o iginal head 20,21 wi h 5 il e s o he ou uni s (see Fig. 2). Each measu emen spo is illumina ed wi h a ligh emi ing diode (LED) wi h a p incipal emission wa eleng h o 470 nm and a luminous in ensi y in he ange o 7.2 cd o 12 cd. Ligh passes h ough an exci a ion band pass il e wi h a cen e wa eleng h o ≈470 nm and a band pass o ≈40 nm, blocking ligh om he LED wi h wa eleng hs longe han ≈490 nm. Ligh is hen e lec ed o o a long pass dich oic mi o wi h a cu -o wa eleng h o ≈495 nm, and ocused by a lens wi h a ocal leng h o ≈3.1 mm, a nume ical ape u e o ≈0.68 and an an i e lec i e coa ing in he ange o ≈350 nm o ≈700 nm. The emi ed luo escence (F) is collima ed by he same lens, passing h ough he dich oic mi o . The esidual blue ligh is supp essed by a long pass emission il- e wi h a cu -o wa eleng h o ≈510 nm, and luo escence is cap u ed by he con en ional silicon pho odiode wi h a adi- an sensi i e a ea o ≈7.5 mm. 2 The c oss sec ion schema ic o he handheld PCR sys em illus a ing he op ical pa h is shown in Fig. S1 (ESI†). The esul ing pho ocu en is con e ed in o ol age using an ul a-low bias cu en ope a ional ampli ie wi h dielec ically-isola ed ield e ec ansis o inpu s (diFET) as a ansconduc ance ampli ie . In his con igu a ion, he ou sample sys ems sha e op ical il- e s. Fou LEDs a e moun ed in wo pai s. Wi hin each pai , he LEDs we e pa allel, hus equi ing only wo exci a ion and wo dich oic il e s o all 4 LEDs. Finally, he e is only a single emission il e o all pho odiodes. A po en ial expan- sion o eigh sys ems would equi e addi ional LEDs and pho odiodes wi h ampli ie s. 2. Con ol elec onics A p e iously published sys em 18 had one lock-in ampli ie o a single luo escence de ec ion sys em and a second one o empe a u e measu emen . This scena io was e y ine icien since luo escence was moni o ed o ≈2 s du ing each PCR cycle. The second lock-in ampli ie o empe a u e measu e- men was used du ing he en i e PCR ope a ion. Ou cu en sys em employs a single lock-in ampli ie o moni o he sam- ple empe a u e and cap u e luo escence om all ou spo s. The sys em hea e s we e connec ed in a se ial–pa allel combina ion, whe ein he sys em con olled he a e age em- pe a u e o all ou hea e s. We used a simila AC biased Whea s one b idge o con e he esis ance o he esis ance empe a u e de ec o (RTD) in o a DC ol age as p e iously desc ibed. 18 The con ol elec onics o he op ical sys em was a simpli- ied e sion o ou p e ious wo k. 18 He e, each PCR sys em (spo ) had i s own LED, collima ing lens and a pho odiode wi h a espec i e ansconduc ance ampli ie while he op i- cal il e s a e sha ed. We ac i a ed one LED a a ime, he eby eeding he signal o a single, co esponding ans- conduc ance ampli ie . Ou pu s o he ou ampli ie s we e connec ed oge he and p ocessed as one signal. The com- ple e schema ic o he PCR sys em is shown in Fig. S2 (ESI†). The inciden pho ocu en s om he pho odiodes we e con e ed in o ol age using ou dedica ed ope a ional am- pli ie s. The ampli ie ou pu s we e connec ed oge he and a single composi e signal was u he p ocessed. The c oss alk be ween ou measu ed spo s was minimized since one LED was ac i a ed a a ime; he e o e he esul ing o al ampli- ude o he p ocessed pho ocu en o igina ed om a single dedica ed PCR eac ion. All impo an de ices a e lis ed in Table S1 (ESI†). Expe imen al A ypical eal- ime PCR p o ocol wi h an in e cala ing dye such as SYBR-G een I ini ia es wi h a ho s a o ac i a e he polyme ase. PCR cycles consis o dena u a ion, annealing and ex ension s eps. The luo escence ampli ude is measu ed a he end o he ex ension s ep o a pe iod o ≈2 s. We con- olled he hea e empe a u e using he p opo ional in eg a i e-de i a i e (PID) closed eedback loop me hod. The amoun o hea deli e ed h ough dissipa ed Joule hea ing was con olled using a pulse-wid h modula ion (PWM) ech- nique. The las ≈2 s o he ampli ica ion cycle we e used o Fig. 2 Schema ic illus a ion o he in eg a ed op ical head. Blue a ows show he op ical pa h om one LED wi h il e s o he VRC. The g een a ow shows he op ical pa h o exci ed luo escence o he pho odiode. Ligh emi ed om a blue LED passes h ough he blue il e in o de o emo e he g een po ion o emi ed ligh , hen e lec s o o he dich oic mi o h ough a donu -shaped hea e and is ocused on he sample by an asphe ical lens. Exci ed luo escence is collima ed by he same lens, passing h ough he dich oic mi o wi h he blue po ion il e ed ou by a g een il e . Residual g een ligh in- e ac s wi h a pho odiode and induces a pho ocu en , which is u he p ocessed. Lab on a ChipPape Open Access A icle. Published on 16 Decembe 2015. Downloaded on 23/06/2016 07:07:56. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online Lab Chip,2016,16,586–592 | 589This jou nal is © The Royal Socie y o Chemis y 2016 luo escence moni o ing (see Fig. 3). In his s ep, ollowing empe a u e s abiliza ion, we moni o ed he du y cycle o he PWM and calcula ed i s a e age. Du ing he las ≈2 s, he eedback loop was disconnec ed, he empe a u e was no moni o ed and he a e age alue o he PWM was employed. Du ing he sys em de elopmen phase, we moni o ed he hea e empe a u e and ound ha he me hod desc ibed abo e gi es us a empe a u e a ia ion o less han ±0.5 °C o e a ≈2 s ime in e al. The sample empe a u e ollows he hea e empe a u e wi h a ≈1.5 s delay, 22 he e o e a ±0.5 °C empe a u e a ia ion a he hea e does no a ec he PCR pe o mance. The measu ed empe a u e p o ile om ≈6 PCR s eps is shown wi hin he sys em liquid c ys al dis- play (LCD) in Fig. 4A. Du ing he las wo seconds o he ex ension s ep, he luo escence measu emen sys em was ac i a ed. Sequen- ially, each LED was indi idually powe ed o ≈0.5 s and he emi ed luo escence was cap u ed by he espec i e pho odi- ode and lock-in ampli ie . A he comple ion o an ampli ica- ion cycle, he sys em was swi ched back in o he empe a u e measu emen mode, ini ia ing he s a o a new cycle. The PCR ampli ica ion cu es we e plo ed o each spo and he cap u ed ampli ude o luo escence was displayed on he LCD display. The ypical PCR ampli ica ion cu e is shown in Fig. 4B. Di ec ly ollowing he PCR p ocess, mel ing cu e analysis (MCA) was pe o med. Since empe a u e measu e- men du ing he MCA is ime consuming, we pe o med his s ep wi hou a eedback loop. In he cou se o he PCR, we moni o ed and eco ded he PWM du y cycle o h ee ixed empe a u e poin s: dena u a ion, annealing and ex ension empe a u es. These h ee poin s we e used o calcula e he equi ed du y cycle o empe a u e scans anging om ≈68 °C o≈94 °C wi hou a closed eedback loop. The sys em was s abilized a ≈68 °C. The empe a u e o each hea e was hen g adually inc eased o ≈95 °C while he luo escence in each o he ou spo s was sequen ially measu ed. The mea- su emen o each spo equi ed a du a ion o ≈0.5 s, hence ≈2 s was equi ed o measu e all 4 VRCs. This measu emen se up allowed o luo escence measu emen om each spo wi h an o se o ≈0.25 °C be ween adjacen VRCs. Once he MCA was comple ed, we s abilized he sample a he as- sumed empe a u e o ≈95 °C and hen measu ed he ac ual empe a u e. Di ec ly ollowing his, he sys em's cen al p o- cessing uni (CPU) pe o med wo co ec ions. Fi s , he MCA was ecalcula ed based on he ac ual inal empe a u e. Sec- ond, he co ec ion accoun s o he empe a u e o se be- ween indi idual VRCs. Consequen ly, he MCAs o each spo we e ecalcula ed acco dingly. Finally, a nega i e de i a- i e alue o luo escence wi h espec o empe a u e (−dF/ dT) was calcula ed. Fig. 3 PCR he mal p o ile o a single ampli ica ion s ep. The hea e empe a u e ( ed pa ) is linea ly p opo ional o he buil -in lock-in ampli ica ion ou pu , which was cap u ed wi h an oscilloscope. While pe o ming dena u a ion a ≈93 °C, annealing a ≈56 °C and mos o he ex ension s ep a ≈72 °C, he buil -in lock-in ampli ie is u ilized o measu e he a e age empe a u e o all ou hea e s. In he las wo seconds o he ex ension s ep, he lock-in ampli ie is used o sequen- ially p ocess he luo escence signal (g een pa ) om he 4 measu e- men spo s (ci cled a ea). These da a a e s o ed in hei o iginal o ma in analog- o-digi al con e e uni s (ADC uni s) wi hin he memo y o a mic ocon olle . Once luo escence is measu ed, he hea e is powe ed by he a e age alue o pulse wid h modula ion ob ained du ing he ex ension phase. Fig. 4 (A) An assembled PCR sys em wi h ou VRCs, each consis ing o a ≈0.5 μL sized sample co e ed wi h ≈1.5 μL o M5904 mine al oil, showing he PCR empe a u e p o ile (p o ocol) wi hin he LCD display. The de ice size is 82 mm ×45 mm ×20 mm (leng h, wid h and heigh ). Scale ba is 40 mm. The p o ocol s a ed by a “ho s a ” o ac i a e he polyme ase enzyme o ≈10 min a ≈95 °C, ollowed by 40 cycles o PCR ampli ica ion. Each ampli ica ion cycle consis ed o 3 s eps. Fi s , dena u a ion o ≈10 s a ≈95 °C, hen annealing o ≈10 s a ≈50 °C and he las s ep was ex ension o ≈15 s a ≈68 °C (ins ead o ypical ≈72 °C), du ing which luo escence was measu ed. The LCD display shows 6 cycles. A he comple ion o each PCR ampli ica ion cycle, luo escence ampli ude a each spo was calcula ed and ampli ica ion cu es we e plo ed in (B). We placed NTC a posi ion 1, a low concen a ion o complemen a y DNA (cDNA) om H7N9 HA gene a posi ion 3, a medium concen a ion a posi ion 2 and he highes concen a ion (posi i e con ol) a posi ion 4. The esul s show ha he PCR eac ion was success ully accomplished wi hou PCR ampli ica ion o he NTC sample. Fu he mo e, he esul s p o e ha he samples we e no c oss con amina ed, he eby elimina ing alse posi i e ou comes. Posi i e con ol esul s a posi ion 4 indica e he absence o alse nega i e esul s, he eby showing a success ul PCR ampli ica ion p ocess. Lab on a Chip Pape Open Access A icle. Published on 16 Decembe 2015. Downloaded on 23/06/2016 07:07:56. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online 590 |Lab Chip,2016,16,586–592 This jou nal is © The Royal Socie y o Chemis y 2016 The de ice pe o mance was e alua ed using syn he ic complemen a y DNA (cDNA) o he hemagglu inin o he H7N9 a ian in luenza i us. Fo wa d and e e se p ime s we e chosen as sugges ed ea lie : 23 o wa d p ime : TACAGGGAAGAGGCAATGCA, e e se p ime : AACATGATGCCCCGAAGCTA, gi ing a o al amplicon leng h o 104 base pai s wi h a mel ing empe a u e o ≈81.1 °C, as measu ed using a comme cial eal ime PCR sys em. The PCR mas e mix was p epa ed by mixing ≈2μLo Fas S a DNA Mas e SYBR G een I, ≈2μL o MgCl 2 solu ion, ≈2μL o sample HA (5 ×10 −5 ng μL −1 ), ≈0.3 μLo ≈400 mg mL −1 BSA solu ion and p ime s in a inal concen a ion o ≈1.8 ×10 −6 mol L −1 . We added deionized (DI) wa e wi h a e- sis i i y highe han 18 MΩcm a 25 °C o c ea e he inal olume o ≈20 μL. The NTC sample had ≈2μL HA gene ol- ume eplaced wi h DI wa e . Fi s , we pe o med basic eal- ime PCR wi h di e en con en s o cDNA pe μL as shown in Table 1 wi h NTC a po- si ion 1 and di e en con en s a posi ions 2 o 4. The ampli- ica ion cu es a e shown in Fig. 5A. Once he PCR p ocess was comple ed, we also conduc ed a MCA (no shown he e). The MCA shape is no sui able o pe o ming high- esolu ion analysis; 24 ne e heless, i does show ha a spe- ci ic DNA was ampli ied wi h he mel ing empe a u e o 83.36 ± 0.63 °C (mean ± s anda d de ia ion), in close p oxim- i y o he measu ed alue o T M ≈81.1 °C. The ma ginal di - e ence in he T M alues is due o he unce ain y o calib a- ion p ecision o he comme cial PCR sys em used as a benchma k ool. The T M esolu ion is su icien o e i y spe- ci ic DNA ampli ica ion; howe e , i may no be sui able o pe o ming high- esolu ion mel ing cu e analysis. 25 We hen pe o med a se ies o ou iden ical measu e- men s using an HA con en o ≈5×10 −5 ng in a μL o cDNA (see da a in Table 2 and a g aphical ep esen a ion in Fig. 5B). Di e en PCR loca ions esul ed in pe o mance a - ia ion, consequen ly p oducing mean C T alues in he ange o ≈8 o≈9.6 wi h a s anda d de ia ion anging be ween ≈0.8 and ≈1.5. The di e ence in C T alues a posi ions 2 o 4 migh be caused by impe ec ions due o manual VRC place- men . The VRC a posi ion 1 appea s o ha e a lowe hea ans e a e han VRCs a o he posi ions. We a ibu e he a ia ion o a slowe ansi ion om ampli ica ion o sa u a- ion o he DNA ampli ica ion cu e (Fig. 5B). We u he p e- sume ha hea e de ec s a posi ion 1 could gi e ise o a empe a u e ha is di e en in compa ison wi h he o he h ee posi ions, consequen ly leading o a di e ing PCR e iciency. Addi ionally, his disc epancy could be a ibu ed o he s ess induced du ing chip- o-PCB solde ing, gi ing ise o bending o he chip. The silicon chip de o ma ions could cause bo h a ia ions in he in e media e oil laye hickness and a di e ing hea a e. Finally, we ob ained s anda d PCR cu es. The samples we e p epa ed by he ollowing p ocedu e. We mixed a sam- ple wi h cDNA co esponding o 12 500 copies pe 200 nL ol- ume. This sample was dilu ed 10×, yielding 1250 copies pe 200 nL olume; he nex dilu ion yielded 125 copies pe 200 nL olume. The las wo dilu ions had a s a is ical numbe o 12.5 copies pe 200 nL and 1.25 copy pe 200 nL olume, e- spec i ely. The PCR esul s as well as he no malized da a a e shown in Fig. 5C and D, demons a ing ha ou po able eal- ime PCR is able o de ec a single DNA copy wi h an ex- cellen e iciency o 0.91 ± 0.05 pe cycle (mean ± s anda d de ia ion), which is well wi hin he equi ed ange o PCR e - iciency be ween 0.8 and 1.0. Discussion The PCR p o ocol consis ed o a ≈10 min ho s a a ≈95 °C, ollowed by 40 cycles o ≈10 s a ≈95 °C, ≈10 s a ≈50 °C and ≈15 s a ≈68 °C. Once he PCR ampli ica ion was com- ple ed, we conduc ed an MCA wi h a scan a e o ≈0.2 °Cs −1 . This p o ocol equi ed a o al ime o ampli ica ion o less han 35 minu es wi h an addi ional ≈150 s o he MCA. The ul ima e speed o he PCR was no he p ima y a ge o his wo k. Ne e heless, he ime equi ed o DNA ampli ica ion can be sho ened by using di e en ypes o ho s a s, Taqman chemis y (o bo h) 19 o e en no using he ho s a a all. 26 He e we used he same hea e as in ou p e ious wo k. The dissipa ed Joule hea Pdepends on he squa e o ol age V: P=V 2 /R,whe eRis he hea e esis ance. The Joule hea dissi- pa ion and he consequen hea ing a e we e enhanced wi h ei he an inc eased ol age bias o by lowe ing he hea e esis- ance. In a p e ious wo k, 22 we inc eased he bias ol age up o 20 V using an ex e nal powe supply. He e, he hea e is powe ed using a 12 V ex e nal powe supply in ei he an AC/DC con e e o a ba e y ype con igu a ion. A change in his ol age would equi e addi ional space o a s ep up ol - age con e e . The addi ional ea u e would u he equi e ei he a edesign o he hea e on a mic omachine, equi ing a new mask o he me al li hog aphy le el, o he use o a hicke me al laye wi h a lowe shee esis ance. Bo h cases would equi e he ab ica ion o new PCR chip a chi ec u es. 14 The cu en PCR chip layou is shown in Fig. S3 (ESI†) and he chip ab ica ion p ocess in sec ion 5 (ESI†). In p inciple, he undamen al limi a ion in he speed o he de ice is de e mined by he hea ans e be ween he hea e and he sample, which is ≈1.5 s o each empe a u e s ep. Ne e heless, he de ice can s ill un as as as i s p ede- cesso achie ing ≈9.5 s pe PCR cycle, s ill being conside ed as one o he as es eal- ime PCRs demons a ed a ha poin in ime. 19 Table 1 Typical esul s wi h NTC (posi ion one) se ing as nega i e con- ol and h ee di e en sample concen a ions a posi ions 2 o 4. The sample a posi ion 4 se es as posi i e con ol Posi ion Mean C T S anda d de ia ion Concen a ion HA (ng μL −1 ) 1—— — 2 28.7 1.5 ≈5.0 ×10 −8 3 27.0 1.0 ≈7.5 ×10 −8 4 20.0 1.0 ≈5.0 ×10 −6 Lab on a ChipPape Open Access A icle. Published on 16 Decembe 2015. Downloaded on 23/06/2016 07:07:56. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online Lab Chip,2016,16,586–592 | 591This jou nal is © The Royal Socie y o Chemis y 2016 Fu he mo e, we enhance he sys em obus ness by no in- co po a ing mo ing pa s. The sys em ligh sou ce consis s o 4 LEDs wi h an es ima ed li e ime o mo e han 50 000 hou s. A single 40 cycle PCR un equi es each LED o ope a e o less han 1 min. The mos ulne able pa is he mic omachined silicon chip moun ed di ec ly on he main PCB. We en ision a new e sion o ou sys em, cu - en ly unde de elopmen , wi h he b i le silicon chip moun ed on a dedica ed PCB. The e o e, i he agile pa is damaged, a eplacemen silicon chip can be easily ex- changed. In o de o limi in e e ence be ween PWM pulses and empe a u e sensing signals, he layou o he mic o- machined silicon chip will inco po a e elec ical shielding be ween in eg a ed hea e s and senso s. In his scena io, he chip size will inc ease o ≈18 mm ×18 mm. Also, his con igu a ion p o ides addi ional space in o de o u he modi y he op ical housing and acili a e he emo al o he PCB om he op ical pa h. The cu en e sion o he sys em exhibi ed a la ge sel -induced luo escence due o he PCB being illumina ed by he blue LEDs. Finally, we plan o educe he complexi y o he op ical housing by educing he numbe o pa s. This would also allow us o eplace he silicon chip once a ia ions in PCR e iciency a e disco e ed. Fig. 5 (A) Single PCR un as i appea s in he PCR display. These PCR ampli ica ion cu es show esul s om he 4 posi ions o he PCR de ice. We used complemen a y DNA (cDNA) om he H7N9 HA gene o es ing pu poses. AU s ands o a bi a y uni s. Once he PCR was comple ed, he MCAs we e pe o med wi h a mel ing empe a u e alue o 83.36 ±0.63 °C (mean ±s anda d de ia ion). Measu emen unce ain ies emana e om he manual placemen o he d ople . Sligh d ople misalignmen s a he hea e cause empe a u e a ia ions be ween a ious expe imen al uns, he eby a ec ing he o e all PCR e iciency. Also, due o hese empe a u e a ia ions, we eadily obse e a sligh shi in he measu ed mel ing empe a u e. (B) Measu emen s pe o med a he 4 posi ions wi h iden ical concen a ion o all samples pe o med h ee imes o supp ess andom e o . The co esponding ex ac ed c i ical h esholds (C T ) a e shown in Table 2. A g ea e alue o C T a posi ion 4 sugges s a lowe ampli ica ion e iciency a ha posi ion. This may be caused by a empe a u e a ia ion due o a non-op imized bonding p ocess o he sili- con chip o he PCB. (C) PCR esul s om posi ion 1 wi h he calcula ed numbe o cDNA copies in he sample om ≈12 500 down o ≈1.25. These numbe s a e calcula ed by a 10×dilu ion s a ing om ≈12500. Since only whole numbe s o cDNA copies pe sample exis , ac ional alues imply he s a is ically mos p obable alue. Each expe imen was pe o med h ee imes. (D) Ex ac ed s anda d eal- ime PCR cu e om esul s in Fig. 5C showing he C T alue as a unc ion o LOG (cDNA concen a ion). The slope o −3.02 ±0.16 cycles a h eshold pe decade (mean ±s an- da d de ia ion) co esponds o he PCR e iciency o 0.91 ±0.05 pe cycle (mean ±s anda d de ia ion). Table 2 Resul s o c i ical h eshold om 4 measu emen s a each PCR loca ion wi h iden ical concen a ion o he HA gene. The g aphical ou - pu is shown in Fig. 5B. The disc epancy be ween esul s om indi idual samples was p obably caused by sample misalignmen wi h espec o he hea e as hey we e placed manually Posi ion Mean C T S anda d de ia ion Concen a ion HA (ng μL −1 ) 1 9.6 1.5 ≈5.0 ×10 −5 2 9.0 1.2 ≈5.0 ×10 −5 3 9.5 0.6 ≈5.0 ×10 −5 4 8.0 0.8 ≈5.0 ×10 −5 Lab on a Chip Pape Open Access A icle. Published on 16 Decembe 2015. Downloaded on 23/06/2016 07:07:56. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online 592 |Lab Chip,2016,16,586–592 This jou nal is © The Royal Socie y o Chemis y 2016 Fu he mo e, in u u e expe imen s, we plan o use ei he mic oscope glass co e slips wi h up on lyophilized PCR o a single s ep RT-PCR mas e mix. In his con igu a ion, he pipe ed ≈200 nL olume sample will be en i ely composed o DNA (RNA). Conclusions We designed and es ed one o he smalles eal- ime PCR de- ices. I has a leng h o ≈100 mm, a wid h o ≈60 mm and a heigh o ≈33 mm and weighs only ≈90 g. The de ice mea- su ed 4 PCRs simul aneously in less han ≈35 min, including MCA. The sample was p ocessed in he o m o a i ual eac- ion chambe (VRC) whe e 200 nL o a sample was placed on a disposable glass co e slip co e ed wi h mine al oil o p e- en wa e e apo a ion om he sample. The sample only in- e ac s wi h he glass co e slip o elimina e possibili ies o sample- o-sample c oss-con amina ion; he glass elemen was a single-use, disposable sys em componen . Ou nega i e con- ol es s u he demons a e he lack o c oss con amina ion be ween samples. The sys em depic ed in Fig. 4 was success- ully u ilized o a leas 100 dis inc PCR uns. We ha e dem- ons a ed i s pe o mance by ampli ying he cDNA o an HA gene o he H7N9 a ian in luenza i us and displayed he e- sul s on an in eg a ed LCD display. We demons a ed he ca- pabili y o simul aneously unning 4 samples a a ime wi h good ep oducibili y. The PCR e iciency was demons a ed by ob aining a PCR s anda d cu e in he ange o 12500 o 1.25 copies wi h an achie ed slope o −3.02 ± 0.16 cycles a h eshold pe decade (mean ± s anda d de ia ion). The alue co esponds o a PCR e iciency o 0.91 ± 0.05 pe cycle (mean ± s anda d de ia ion). The sys em was also capable o de ec ing a single DNA copy wi hin he sample. The cap u ed da a was subsequen ly ans e ed o a pe - sonal compu e (PC) ia a USB in e ace o u he p ocess- ing. This iny eal- ime PCR de ice is a p omising diagnos ic sys em o emo e clinics as well as a ool o educa ional in- s i u ions demons a ing he powe o a eal- ime PCR as “seeing is belie ing”. The sys em h oughpu can be doubled using a single channel mul iplexing me hod as demons a ed ea lie . 27 Acknowledgemen s P. Neužil acknowledges pa ial inancial suppo om he Cen al Eu opean Ins i u e o Technology (CEITEC), g an numbe CZ.1.05/1.1.00/02.0068. The au ho s g a e ully ac- knowledge he NIST CNST NanoFab s a o help ul discus- sions and assis ance wi h de ice ab ica ion. This a icle iden- i ies ce ain comme cial equipmen , ins umen s, and ma e ials o speci y he expe imen al p ocedu e. Such iden i i- ca ion does no imply ecommenda ion o endo semen by he Na ional Ins i u e o S anda ds and Technology, no does i imply ha he equipmen , ins umen s, and ma e ials iden- i ied a e necessa ily he bes a ailable o he pu pose. 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