PAPER IN FOREFRONT

Consecutive and automatic detection of multi-gene mutationsfrom colorectal cancer samples by coupling droplet array-basedcapillary electrophoresis and PCR-RFLP

Yiming Feng1& Tingting Hu1

& Pan Fang2& Linlin Zhou1

& Wanming Li1 & Qun Fang2& Jin Fang1

Received: 22 January 2020 /Revised: 20 February 2020 /Accepted: 2 March 2020# Springer-Verlag GmbH Germany, part of Springer Nature 2020

AbstractThe efficacy of targeted therapy is associatedwith multi-gene mutation status. Carrying out effective multi-genotyping analysis incombination has been a challenge in clinical settings. We therefore developed a droplet-based capillary electrophoresis (CE)system coupled with PCR-restriction fragment length polymorphism (PCR-RFLP) technology to detect multi-gene mutationsfrom a small volume of samples. A 16 × 16 200-nL droplet array for sample encapsulation was constructed on a glass chip. Theelectrophoresis system consisted of a tapered vertical capillary filled with polyvinylpyrrolidone, a laser-induced fluorescencedetector, and a high voltage power supply. Notably, a droplet-based electrokinetic sample introduction method and a “∩” shapecapillary were developed to facilitate consecutive droplet sampling using a home-made automatic control module. The DL2000DNA marker was consecutively separated, achieving high migration time and plate number reproducibility. The system wasfurther applied to detect PCR-RFLP products. For colorectal cancer (CRC) cell lines,KRAS, BRAF, and PIK3CAwere genotypedwith a sensitivity of 0.25%. For CRC patient specimens, 30 samples were consecutively and automatically multi-genotypedwithout inter-sample contamination, with a lowest mutation frequency of 0.37%. For the first time, we developed a droplet-basedCE system for consecutive DNA analysis with low sample consumption. This automated CE system could be further developedto integrate the full process of gene mutation detection, serving as a more effective platform for individualised therapy.

Keywords Capillary electrophoresis . Droplets . Multi-gene detection . Genemutation . PCR-RFLP

Introduction

Gene mutation is a critical molecular change occurringduring cancer. Notably, it was found that gene mutationis related to the efficacy of cancer treatments such astargeted therapy. Although the introduction of targetedtherapy has shown superior specificity and efficiency to

traditional chemoradiotherapy, only a portion of patientscan benefit from this due to their differentiating genotypes[1, 2]. For example, the KRAS mutation was identified as anegative biomarker for anti-EGFR-targeted therapy [3].Santos et al. [4] reported that the KRAS mutation is asso-ciated with a worse outcome for anti-EGFR-targeted ther-apy compared with wild-type KRAS. The response rates

Yiming Feng and Tingting Hu contributed equally to this work.

Electronic supplementary material The online version of this article(https://doi.org/10.1007/s00216-020-02567-y) contains supplementarymaterial, which is available to authorized users.

* Qun Fangfangqun@zju.edu.cn

* Jin Fangjfang@cmu.edu.cn

1 Department of Cell Biology, Key Laboratory of Cell Biology,Ministry of Public Health, and Key Laboratory of Medical CellBiology, Ministry of Education, China Medical University, No.77Puhe Road, Shenyang North NewArea, Shenyang 110122, Liaoning,China

2 Institute of Microanalytical Systems, Department of Chemistry,Zhejiang University, Chemistry Experiment Building,Hangzhou 310058, Zhejiang, China

https://doi.org/10.1007/s00216-020-02567-yAnalytical and Bioanalytical Chemistry (2020) 412:3037–3049

/Published online: 6 April 2020

among KRAS wild-type and mutant patients were approx-imately 42% and 20%, respectively. Due to the strong cor-relation between gene mutation and resistance to targetedtherapy, mutational detection to identify non-responders ismandatory before administering targeted therapy [5].Further, an increasing amount of studies have suggestedthat variations of other genes also meditate resistance totargeted therapy [2, 6, 7]. In CRC patients, growing evi-dence shows that in addition to KRAS, the genetic status ofBRAF, PIK3CA, and PTEN can also predict the outcome ofanti-EGFR-targeted therapy [8, 9]. Tural et al. [10] inves-tigated the efficacy of anti-EGFR-targeted therapy amongKRAS wild-type patients. When taking BRAF, PIK3CA,and PTEN into consideration, the wild-type subgroup hadan increased response rate of 66% from 25%. The responserate can therefore be significantly increased by performingmulti-genotyping, indicating that multi-gene mutationaldetection is necessary to provide more accurate outcomeprediction. This can consequently avoid ineffective, costly,and toxic treatment for personalised therapy.

An ideal multi-gene mutation analysis method suitable fora clinical setting needs to possess various features. One suchfeature is low sample consumption, enabling the detection ofmulti-gene mutations using a single patient sample. Growingreports show that during targeted therapy, acquired resistancemay occur due to newly produced gene mutations. As such, asmall sample requirement can facilitate multi-gene detectionfor monitoring the response to targeted therapy and regulatingtreatment prescription [8, 11]. Excellent sensitivity would alsobe ideal for analysis, assuring the detection ofminor mutationsin a low sample volume. Further favourable features includefast detection speed and high automation. Although Sangersequencing is currently regarded as the gold standard for genemutation detection, its low detection sensitivity (20–30%)cannot meet current clinical demand for cancer analysis [12].In recent years, next-generation sequencing (NGS) has hadsignificantly increased sensitivity and throughput comparedwith Sanger sequencing. However, NGS can be costly andtime-consuming, and the huge amount of data and analyticalcomplexity does not lend itself well to clinical settings [13,14]. As the gene mutations related to targeted therapy re-sponse are primarily point mutations such as KRAS G12V,BRAF V600E, and PIK3CA H1047R, PCR-based DNA anal-ysis methods are generally applied for genotyping in clinicalpractices. These methods include PCR-single-strand confor-mation polymorphism (SSCP), allele-specific real-time PCR,denaturing gradient gel electrophoresis (DGGE), and PCR-RFLP. These are able to achieve superior detection sensitivityto Sanger sequencing owing to signal amplification by PCR[15–18]. In most cases, gene mutation status is judged byelectrophoretic separation followed by PCR. However, theroutinely used slab gel electrophoresis suffers from both

relatively large initial sample volume and long separationtime, and cannot meet the requirements of multi-genedetection.

To enhance DNA detection speed and sensitivity, severalresearchers have used capillary electrophoresis to replace slabelectrophoresis [19, 20]. CE is a separation technique whichuses a capillary as a separation channel, and higher separationefficiency than that of routine electrophoresis can be achievedby applying a high electric field. CE has been applied to detectDNA by filling the capillary with sieving matrix [21]. Wanget al. [22] identified the p53 mutation from gastric cancertissue by separating the enzyme-digestion products of PCR-RFLP using CE, obtaining superior detection speed and sen-sitivity to slab gel electrophoresis. However, conventionalCE-based DNA separation requires a relatively large initialsample volume of 5–10 μL, similar to slab gel electrophoresis[23]. This large sample consumption might hamper multi-genotyping towards a single or multi-sampling and thereforeanalysis from a given patient.

Droplet-based microfluidic technology can encapsulate re-action solution into independent micro-droplets by manipulat-ing two incompatible liquid phases [24, 25]. Using thedroplet-based sample introduction method, only a nanolitre-scale initial sample was required for CE separation, whichfacilitates multi-sample analysis via high-throughput detec-tion. Niu et al. [26] used a continuous flow droplet chip tointroduce the DNA sample to microchip-based CE separation,and only a 4 nL DNA sample was required for sensitive anal-ysis. However, in most cases, the droplets containing separat-ed components are formed in a continuous flow manner in amicrofluidic chip channel. This easily generates droplets withthe same components, but rarely creates largely different sam-ple droplets [27]. In addition, continuous flow needs to extractaqueous droplets to the separation channel while excludingthe carrier oil. These weaknesses limit the ability of droplet-based CE to perform multiplex sample detection. In our pre-vious study, we developed a two-dimensional droplet arraysystem using a microwell array chip with open features. Asa sampling probe, the capillary was directly inserted into thedroplet to achieve picolitre-scale sample introduction. Thiswas done using the spontaneous sample injection mode with-out the need for the droplet extraction interface [28]. Thissystem was coupled with CE to consecutively separate 25different droplet samples of amino acids. However, the meth-od for introducing a droplet-encapsulated DNA sample forsubsequent CE separation remains challenging. This is be-cause the spontaneous injection method cannot provide suffi-cient force to drive DNA samples into the CE channel filledwith sieving matrix, which is necessary for DNA separation.In this study, we developed an effective droplet-based electro-kinetic injection mode to introduce DNA from nanolitre-scaledroplet samples. We also designed a “∩” shape capillary as a

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sampling probe to facilitate consecutive sampling of differentsamples in the array.

PCR-RFLP is a DNA mutation detection technique inwhich the DNA fragment-covering mutation is PCR-ampli-fied, after which the products undergo an enzyme-digestionreaction. The mutation is detected based on the differencebetween wild-type and mutant genes, according to the numberand size of the enzyme-digestion fragments obtained via elec-trophoretic separation. Due to its advantages of easy operationand accurate detection, PCR-RFLP has been widely appliedfor gene mutation detection in a clinical setting [17, 29]. Weherein used the droplet coupling CE system to separate PCR-RFLP products loaded in a microwell array chip, achievingconsecutive analysis of multi-genes and multi-samples fromCRC patients under an automatic control system. Sensitivityas low as 0.37% was achieved without inter-sample contam-ination by the electrokinetic sample introduction mode.

Materials and methods

Cell lines and specimen samples

The human CRC cell lines CCL-187, HT-29, and HCT116were purchased from American Type Culture Collection(Manassas, VA, USA). The human CRC cell lines RKO andLS174T were obtained from Type Culture Collection of theChinese Academy of Sciences (Shanghai, China). HumanCRC cell lines CCL-187 and RKO were cultivated inDMEM with 10% foetal bovine serum (FBS, Invitrogen,Carlsbad, CA, USA) and 100 U/mL penicillin-streptomycin(Sigma-Aldrich, St Louis, MO, USA). The human CRC celllines HT-29, HCT116, LS174T, and SW480 were cultivatedin RPMI1640 medium containing 10% foetal bovine serumand 100 U/mL penicillin-streptomycin. All CRC cell lineswere cultured in a humid incubator filled with 5% CO2.Formalin-fixed paraffin-embedded (FFPE) tissue sections(5 μm) from 10 CRC patients were obtained from the FirstAffiliated Hospital of China Medical University. The ethicscommittee approved the use of these samples for the purposeof academic research, and all patients who donated samplesprovided written informed consent.

Genomic DNA extraction

The genomic DNA (gDNA) of human CRC cell lines wasextracted using a Genomic DNA Purification Kit (Promega,Madison, WI, USA) according to the manufacturer’s instruc-tions. The concentration and purity of extracted DNA weremeasured using a NanoDrop 2000 (Thermo FisherScientific, Waltham, MA, USA) and adjusted to 50 ng/μLusing deionised water for PCR amplification. Sections ofFFPE 5 μm thick were harvested in a 1.5-mL tube. The

gDNA were extracted using a FFPE DNA Kit (ThermoFisher Scientific) according to the manufacturer’s instructions.The final concentration was also adjusted to 50 ng/μL. Bothcells and FFPE-derived DNAwere stored at 4 °C before use.

PCR-RFLP analysis of PIK3CA, KRAS, and BRAFmutation status

To assess the mutational detection performance of droplet-based CE using the PCR-RFLP technique, we selected thefollowing commonly mutated hotspots in three anti-EGFR-targeted therapy-related genes: KRAS G12V in exon 2;BRAF V600E in exon 15; and PIK3CA H1047R in exon 20.The sequences of each primer set for PCR amplification wereas follows:

KRAS forward primer: 5′-GACTGAATATAAACTTGTGGTAGTTGGACCT-3′.KRAS reverse primer: 5′-CTATTGTTGGATCATATTCGTCC-3′.BRAF forward primer: 5 ′-TCATAATGCTTGCTCTGATAGGA-3′.BRAF reverse primer: 5′-GGCCAAAAATTTAATCAGTGGA-3′.PIK3CA forward primer: 5′-GGAGTATTTCATGAAACAAATGAATGATGCG-3′.PIK3CA reverse primer: 5′-GAGCTTTCATTTTCTCAGTTATCTT-3′.

The 25 μL PCR reaction mixture for all three genescontained 0.2 mM dNTPs, 0.5 mM of each primer, 1× PCRbuffer, 2.5 U rTaq DNA polymerase (Takara Bio, Kusatsu,Japan), and 2 ng/μL gDNA. Amplification was carried outin a T100 Thermal Cycler (Bio-Rad), followed by pre-heating at 94 °C for 5 min. The following cycles were thenperformed for the three genes: 30 cycles at 94 °C for 30 s,65 °C for 1 min, and 72 °C for 1 min (KRAS); 30 cycles at94 °C for 45 s, 60 °C for 30 s, and 72 °C for 30 s (BRAF); and30 cycle at 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 45 s(PIK3CA). Finally, 72 °C for 10 min for all three genes. Afterthermal cycling, RFLP was carried out. The three ampliconsKRAS (107 bp), BRAF (224 bp), and PIK3CA (126 bp) weresubjected to corresponding restriction endonucleases, bywhich the wild-type and mutant alleles of each gene werecleaved into distinct fragments. For the wild-type KRASamplicon, the 107 bp fragment could be digested into 30 bpand 77 bp fragments, while the mutant type remained at107 bp. The 224 bp of wild-type BRAF amplicon could becleaved into three fragments of 13 bp, 87 bp, and 124 bp,while the mutant allele yielded 13 bp and 211 bp fragments.The wild-type PIK3CAwas digested into fragments of 30 bpand 96 bp, while the mutant type remained at 126 bp. Therestriction endonucleases MvaI, TspI, and FspI (Thermo

Consecutive and automatic detection of multi-gene mutations from colorectal cancer samples by coupling... 3039

Fisher Scientific) were applied to the KRAS, BRAF, andPIK3CA amplicons, respectively. A 5 μL PCR product wastypically digested with 1 unit of restriction endonuclease in26 μL digestion buffer. The digestion mixture for both KRASand PIK3CAwas incubated at 37 °C for 10 min. The digestionmixture of BRAF was incubated at 65 °C for 10 min. Afterdigestion, equivoluminal digestion product and 20× Eva green(Bio t ium, Fremont , CA, USA) were mixed fo relectrophoresis-based fragment analysis.

Setup of droplet-based CE system

This system consisted of two modules: the automatic dropletarray module and the CE module (Fig. 1). The automaticdroplet array module included a microwell array chip for sam-ple loading and an automatic x-y-z translation stage modifiedfrom a commercialised CNC engraving machine (Amo01,Amo Electronic Tech, Dongguan, China) for moving the chipprecisely during analysis. The microwell array chip was fab-ricated from a chromium glass plate coated with AZ

photoresist (Shaoguang, Changsha, China) using traditionalphotoetching and wet-etching methods. Briefly, a pre-designed photomask (Kaisheng, Shanghai, China) with a16 × 16 spot array (0.5 mm in diameter, 1 mm between cen-tres) was aligned to the chromium glass plate. The plate wasthen subjected to UV and developed by 0.2 M NaOH.Following this, chromium etching solutionwas used to exposethe unprotected glass layer, which was later processed by wet-etching solution for 10 min to create a 16 × 16microwell arrayon the chip. A glass frame was drilled and then glued to thechip with epoxy adhesive for oil containment. Finally, theplatinum electrode was fixed to the chromium surface on theedge of the chip using conductive adhesive. The microwellarray chip was fixed on the automatic x-y-z translation stage.

The CE module consisted of a capillary (ID 50 μm, OD375 μm, Refine Chromatography, Yongnian, China), a home-made confocal laser-induced fluorescence (LIF) detector foranalyte detection, and a high voltage power supply (DongwenHigh Voltage Co., Tianjin, China) for providing power duringinjection and separation. The home-made confocal LIF detec-tor was modified from our previous compact hand-held LIFdetector [30]. Briefly, we adopted a confocal optical configu-ration which consisted of a 450-nm laser diode, a 450-nmband-pass filter, a dichroic mirror, a collimating lens (NA =0.3), a 525-nm band-pass filter, a mirror, and a photomultipliertube (PMT). A 16-cm-long capillary with an effective separa-tion length of 2.5 cmwas used as a separation column, and theinlet end was carefully ground with the procedure as we re-ported previously [31]. Briefly, first approximate 5 mm pro-tective layer from inlet end was removed. To avoid the block-age from particles produced during the grinding procedure,the capillary inlet was pre-blocked by the solid paraffin.Then, the capillary was mounted on a hand-held drill. Next,the tip was carefully ground on various sandpapers in differentgrids. Finally, the tip was polished by cerium oxide polishingpowder. This tapered tip can facilitate the electrokinetic injec-tion from the droplet. The tapered capillary was bent andvertically fixed on a supporter in a “∩” shape, and both inletand outlet ends were adjusted to the same level. Two slotted500-μL tubes, serving as buffer and waste vials, were fixatedon the x-y-z translation stage and the front supporter, respec-tively. Each vial was equipped with a platinum electrode todeliver high voltage. Automation of consecutive analysis wasachieved by using a LabVIEW written program controllingthe x-y-z translation stage, high voltage power, and confocalLIF-CE.

Separation condition optimisation of droplet-basedCE system

The capillary surface modification and sieving medium con-centration were investigated to find the optimal separationconditions. Firstly, for inner surface modification, three

Fig. 1 Schematic diagram of droplet-based CE system. A Sample injec-tion from the droplet. The capillary inlet end was inserted into the droplet,and DNA was introduced into the capillary by applying high voltagebetween the chip and anode. B Sample separation. To conduct CE sepa-ration of the injected DNA sample, the translation stage re-located thebuffer vial to immerse the capillary

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treatments of 2-(4-morpholino)ethanesulfonic acid (MES)-Tris buffer, 1M HCl, and deionised water were studied.Separation performance was evaluated using a DL2000DNA marker consisting of 6 fragments (100 bp, 250 bp,500 bp, 750 bp, 1000 bp, and 2000 bp) as a model. MES-Tris buffer was made by dissolving both MES and Tris(Sigma-Aldrich) into deionised water to final concentrationsof 80 nM and 40 mM, respectively. Both vials and the capil-lary were filled with 3% (w/v) polyvinylpyrrolidone (PVP),serving as both an electrolyte and sieving matrix. Capillarysurface modification was carried out by flushing the capil-laries for 10 min with MES-Tris buffer, or 1M HCl, ordeionised water. After modification, the capillaries were filledwith 3% PVP sieving medium, and 5 min pre-electrophoresiswas carried out to stabilise the baseline. After that, 200 nLderived DL2000 DNA Marker was dispensed onto the wellsof the chip using a micropipette. The chip was pre-coveredwith mineral oil.

Automatic electrophoretic injection and separation wasachieved by moving the x-y-z translation stage to carry thechip to a position where the targeted droplet was directly be-low the tip of the capillary. The tip of the inlet was theninserted into the droplet through the mineral oil layer via au-tomatic elevation of the x-y-z translation stage. Electrokineticinjection was then carried out by applying 1600V between thechip and the anode. The injection time and current were 2 sand 6 μA respectively. After injection, the x-y-z translationstage re-targeted the buffer vial to the inlet of the capillary.Finally, as soon as the inlet was immersed in the PVP sievingmedium, separation was carried out by applying 1600 V be-tween both vials.

For optimising the sieving medium concentration, 1–4%(w/v) PVP sieving medium was made by dissolving variousamounts of PVP powders (1300000; Sigma-Aldrich) into 1×TBE solution (Sangon Biotech, Shanghai, China). Afteradding the powder, the solution was vortexed for 2 minfollowed by ultrasound for degassing. The MES-Tris-processed capillaries filled with various concentrations ofPVP sieving medium were used to separate the DL2000DNA marker according to the above procedure. The platenumber and migration time were investigated.

Analysis of reproducibility of droplet-based CE system

The separation reproducibility of the droplet-based CE systemwas evaluated by consecutive separation of the DL2000 DNAmarker-loaded droplets. A 200 nL derived DL2000 DNAmarker was dispensed on the different wells of the chip toform a droplet array. For single droplet analysis, three consec-utive sample introductions and separations were carried outfrom a single well. For multi-droplet analysis, seven dropletsfrom different wells were separated consecutively. For boththe single and multi-droplet assays, the migration time and

plate number for the 100 bp fragment of the DL2000 DNAmarker was used to investigate the separation reproducibilityof the system.

Detection sensitivity of droplet-based CE

The mutational detection sensitivity of droplet-based CE wasassessed via PCR-RFLP using PIK3CA H1047R as a model.First, two plasmids containing the 126 bp wild-type and mu-tant PIK3CA gene were constructed using a TA cloning kit(Takara Bio). The plasmids were then extracted and purifiedfrom E. coli DH5α with a Plasmid Mini Kit (QIAGEN,Hilden, Germany). Serially diluted mutant plasmids contain-ing the PIK3CAH1047Rmutation were mixed with wild-typeplasmid to form 8 mixtures at increasing ratios of 1:1, 1:5,1:10, 1:100, 1:200, 1:300, 1:400, and 1:500. These mixtureswere used as templates for the subsequent PCR-RFLP reac-tion. After digestion, 200 nL of each derived product waspipetted onto the chip, then being directly injected for theCE separation with the injection time of 10 s and the currentof 6 μA. All the samples were analysed separately.

Consecutive detection of multi-gene mutationsfrommultiple colorectal cancer cell lines and multipleclinical samples using droplet-based CE system

To evaluate the applicability and reliability of performingmul-tiple mutational detection in combination with the present sys-tem, three anti-EGFR-targeted therapy-related genes wereconsecutively genotyped from both CRC cell lines andFFPE tissue samples. The gDNA from six CRC cell lines,SW480, RKO, HT-29, HCT-116, CCL187, and LS174T, wereextracted. Two hundred nanolitre of each PCR-RFLP productswas pipetted on the wells of the chip to form a droplet array,then being directly injected for the CE separation with theinjection time of 10 s and the current of 6 μA. Their mutationstatus in KRAS G12V, BRAF V600E, and PIK3CA H1047Rwere detected by consecutive separation. Two different con-secutive analysis formats were followed, including consecu-tive genotyping of six single genes from six cell samples andthree different genes from two cell samples. Further, to inves-tigate the clinical applicability, we analysed the results of con-secutive detection of theKRAS,BRAF, and PIK3CAmutationsfrom the FFPE tissue sections of 10 CRC patients. Two hun-dred nanolitre of each PCR-RFLP product was pipetted ontothe chip to form a droplet array. In total, 30-sample consecu-tive genotyping was carried out without replenishing the siev-ing matrix.

Sanger and clone sequencing

The gene status of both CRC cell lines and clinical samplesshowing a mutant peak were confirmed by Sanger sequencing

Consecutive and automatic detection of multi-gene mutations from colorectal cancer samples by coupling... 3041

using a 3730xl DNA Analyser (Sangon Biotech, Shanghai,China). Moreover, for clinical samples showing a mutant peakvia CE but no detectable peaks via Sanger sequencing, moresensitive TA clone sequencing was applied to further confirmthe presence of minor mutations. M13 universal primers wereused for TA clone sequencing.

Results

Separation condition optimisationof the droplet-based CE system

To enhance separation efficiency, the separation conditionswere optimised via capillary surface modification and the con-centration of the PVP sieving matrix. The inner surfaces of thecapillaries were treated with MES-Tris buffer, 1M HCl, anddeionised water separately. Following this, we conducteddroplet-based sample introduction and DL2000 DNA markerseparation to evaluate separation efficiency. As shown inElectronic Supplementary Material (ESM) Fig. S1A, whenthe capillary was treated with 1M HCl, baseline separationsof six DNA fragments were achieved at the first sample intro-duction. However, when consecutive separation reached thesixth sample introduction, there was a sharp decrease in reso-lution. Larger DNA fragments were merged and thereforecould not be distinguished. Thus, replenishing the sievingmatrix medium is needed to restore separation resolution.The MES-Tris-treated capillary not only achieved comparableresolution to that of the HCl-treated capillary for the first sep-aration, but also maintained a similar resolution and migrationtime until the 20th consecutive separation. This good repeat-ability indicated that MES-Tris results in a more stable anddurable inhibition of the electroosmotic flow. In contrast, thedeionised water-treated capillary had poor resolution even forthe first separation, although the treatment procedure was sim-ple. MES-Tris was therefore chosen for the surface treatmentin this study.

A suitable sieving medium is needed for CE separation ofDNA. PVP was therefore chosen, and the concentration wasoptimised. The effect of 1–4% PVP on CE separation isdisplayed in ESM Fig. S1B. All six fragments of theDL2000 DNA marker were clearly resolved using variousconcentrations. As the PVP concentration increased so diddetection time, although the separation resolution increasedaccordingly. Furthermore, higher concentrations of PVP pos-sessed greater viscosity, resulting in difficulty with mediumpreparation and filling. We also created a vertical samplingintroduction method facilitating DNA analysis from the drop-lets. Their efficiency was possibly weakened by the highlyviscous medium due to sampling resistance. Based on theabove information, 3% PVP was selected as the optimalconcentration.

Analysis of reproducibility of droplet-based CE system

To assess the separation reproducibility of the droplet-basedCE system, the droplet array containing the DL2000 DNAmarker was consecutively separated. Figure 2A showed theelectrophoretogram of three consecutive separations from asingle droplet. Baseline separation was achieved for all sixfragments. The RSD of the migration time and plate numberfor the 100 bp fragment are 0.5% and 4.5%, respectively. Thisindicated that the CE system is able to carry out consecutivesampling and separate a small volume of sample from a singledroplet. Further, the consecutive separation of multiple sam-ples from different wells on the chip was performed by auto-matically sequentially sampling and separating seven drop-lets. Figure 2B shows that all peaks are clearly resolved inall seven consecutive separations. The RSDs of the migrationtime and plate numbers are 3.2% and 6.9%, indicating that theCE system is able to consecutively and recurrently separatethe samples from different droplets.

Analysis of detection sensitivity of droplet-based CEsystem

To evaluate the mutational detection sensitivity of the presentsystem, the model samples carrying various amounts of thePIK3CA H1047R mutation were constructed. This was doneby mixing the wild-type PIK3CA plasmid with a serial oflower ratio of the mutant. Samples were subjected to PCR-RFLP and their gene status was detected by the droplet-based

Fig. 2 Reproducibility assay of consecutive analysis from 200 nLdroplet. A The electropherogram of three-sample consecutive analysisfrom one droplet. B The electropherogram of seven-sample consecutiveanalysis from 7 droplets on different wells

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CE system. As shown in Fig. 3B, a significant 126 bp mutantpeak could be observed at a mutation frequency of 1:10.Although the mutant signals were weakened and mutationfrequency decreased, the mutant peak was still detectablewhen the ratio dropped to 1:400. This indicated that the mu-tational detection sensitivity of the droplet-based CE systemwas 0.25%. This sensitivity is superior to the Sanger sequenc-ing (1:4) method shown in Fig. 3C.

Validation of droplet-based CE system usingPCR-RFLP

To investigate the ability of PCR-RFLP to accurately resolvedifferent gene mutations derived from cell samples, six CRCcell lines were used to test gene status. First, genotyping of

PIK3CA was carried out. As shown in Fig. 3A, the enzyme-digested fragments of HT-29 and SW480 cells showed twopeaks (30 bp, 96 bp), suggesting they carry wild-typePIK3CA. Three peaks appeared on the electropherogram forLS174T, HCT-116, CCL-187, and RKO cells (30 bp, 96 bp,and 126 bp), indicating the presence of the mutant allele. Theresults obtained from the droplet-based CE system were con-sistent with previous literature [32]. Further, we also conduct-ed genotyping ofKRAS and BRAF. The gene status ofKRAS isdisplayed in ESM Fig. S2A. RKO, HT-29, and HCT-116 cellswere identified to be wild-type, with two peaks on the elec-tropherogram (30 bp, 77 bp). SW480 cells displayed only asingle peak (107 bp), suggesting the presence of mutantKRAS. CCL-187 cells displayed three peaks (30 bp, 77 bp,and 107 bp), indicating the presence of heterozygous mutant

Fig. 3 Detection of PIK3CAmutation in 6 CRC cell lines anddetection sensitivity of droplet-based CE system. A Genotypingof six CRC cell lines with knownPIK3CA gene status. FspI wasused to digest 126-bp PIK3CAamplified from gDNA, and then200 nL of each product wasanalysed separately. Wild-typePIK3CA shows two peaks, whilethe heterozygous mutant showsthree peaks. B Detection sensitiv-ity of droplet-based CE. The mu-tant PIK3CA plasmid was addedto the wild-type plasmid at an in-creasing ratio and analysed by Bthe present system and C Sangersequencing. The arrows in thegraph mark the mutant peak

Consecutive and automatic detection of multi-gene mutations from colorectal cancer samples by coupling... 3043

KRAS. The results for BRAF are shown in ESM Fig. S2B.LS174T, CCL-187, HCT-116, and SW480 cells resulted inthree fragments, suggesting the presence of the wild-type(13 bp, 87 bp, and 124 bp). RKO cells displayed a mutantBRAF profile with two peaks (13 bp, 211 bp). HT-29 cellsdisplayed four peaks, suggesting the presence of heterozygousmutantBRAF (13 bp, 87 bp, 124 bp, and 211 bp). The status ofboth KRAS and BRAF were also completely in line with pre-vious reports [32]. The high accuracy obtained from all threegenes verifies the capability of the droplet-based CE system toperform genotyping using cell samples.

Ability of droplet-based CE system to performmultiple gene mutation detection in real samples

To investigate the capability of droplet-based CE to detectmulti-gene mutations in combination, PCR-RFLP productsfrom different cell lines were consecutively separated bythe droplet-based CE system for gene status detection with-out replacing sieving medium or washing with buffer.First, single genes from six CRC cell lines were genotyped.KRAS G12V (Fig. 4A (a)), BRAF V600E (Fig. 4A (b)), andPIK3CA H1047R (Fig. 4A (c)) were consecutivelyanalysed in the six cell lines, and the gene mutation statusobtained from the present system was in line with existingliterature [32]. Next, a more complex consecutive analysis

was performed using combinational detection of KRASG12V, BRAF V600E, and PIK3CA H1047R derived fromtwo cell lines. As shown in Fig. 4B, the genotyping diver-sity of all six cell lines was accurately obtained. Theseresults indicated that the droplet-based CE system couldeffectively detect multi-sample mutations without inter-disturbance.

Based on the results of cell line analysis, we used thesystem to analyse real clinical samples. The PCR-RFLPproducts from FFPE sections of ten CRC patients wereseparately loaded into a microwell array chip (threewells/gene for each patient) to construct a droplet array.The samples were automatically and consecutively intro-duced and separated one by one under the control of theLabVIEW program. Table 1 lists the mutation status ofKRAS G12V, BRAF V600E, and PIK3CA H1047R in theFFPE samples identified by present system. The electro-phoretogram is shown in Fig. 5. To confirm genotypingaccuracy, Sanger sequencing was used to verify seven intwelve mutations detected by droplet-based CE (ESM Fig.S3A). Another five mutations were further verified byclone sequencing, with the lowest mutation frequency of0.37% (1/272) occurring for the KRAS gene in sample 10(ESM Fig. S3B). The results indicated that the droplet-based CE system could accurately detect gene mutationwith high sensitivity. This automatic detection system

Fig. 4 Consecutive detection of KRAS, BRAF, and PIK3CA in CRC samples using droplet-based CE system.A Consecutive detection of single gene insix CRC cell lines. B Consecutive detection of three genes in combination in two CRC cell lines

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enabled high-throughput multi-gene detection with lesslabour intervention.

Discussion

Effective and accurate detection of multi-gene mutations isessential not only for monitoring cancer progress but alsofor guiding targeted therapy [33, 34]. Here, a droplet-basedCE system was developed and applied to separate PCR-RFLP-derived DNA products. We achieved consecutiveand automatic analysis of multi-gene mutations from bothCRC cell lines and FFPE tissue sections obtained fromCRC patients with low sample consumption and high de-tection sensitivity.

To enable the detection of multi-gene mutations using asingle clinical sample, a detection method for analysing anappropriately small sample volume and amount of DNA isrequired. Several reports have successfully performed PCRwithin micro-droplets with significant reduction in sampleconsumption [35, 36]. However, traditional slab gel electro-phoresis is difficult to couple with droplet PCR due to therelatively large sample volume required for detection. Here,we established a droplet-based sample introduction method toCE for DNA analysis, achieving nanolitre-scale PCR-RFLPproduct separation and mutation detection. Currently, mostresearchers have used the spontaneous injection method forsample introduction when developing a droplet-based CE sys-tem [28, 37]. However, a viscous sieving matrix is needed forDNA separation. This high viscosity hampers introduction ofDNA into the capillary, leading to low detection sensitivity[38]. Accordingly, we developed a droplet-based electrokinet-ic injection method. Traditional electrokinetic injection usual-ly requires a metal annular tube fixed to the inlet end of thecapillary to connect to the cathode of the high voltage power.However, the annular tube is too big to load and analyse ananolitre-scale droplet sample. In this study, we fabricated

microwells on a chip with a chromium layer instead of a metalannular tube to load droplet samples. This led to the effectiveanalysis of small volume DNA samples with an initial volumeof 200 nL via the electrokinetic injection mode. Because thedroplets were in direct contact with the cathode through thechromium surface, no metal annular tube is required for sam-ple introduction.

Aside from small sample volume, multi-gene mutation de-tection also needs to consecutively analyse a series of dropletswith different contents. In the present study, we fabricated adroplet array to load different DNA samples. To consecutivelyintroduce each sample from the oil-covered droplet array, wedeveloped a vertical sampling method using a capillary as asampling probe. This differs from horizontal sampling, whichhas been used previously by ourselves and others [39, 40].Further, to generate the current circuit required by CE, thesampling capillary was designed in a “∩” shape. The inletend of the capillary was inserted into the droplet, and the outletend was immersed in the buffer vial. Meanwhile, both ends ofthe capillary were adjusted to the same level to prevent theinfluence of gravity during electrophoresis [41]. The droplet-based CE system obtained good reproducibility in terms ofplate number and migration during consecutive analysis ofmultiple droplet samples. Furthermore, we carried out 30 con-secutive genotyping from clinical samples withoutreplenishing fresh matrix between runs. The genotypes fromall the samples were correctly detected indicating the presentsystem can maintain a reproducible separation efficiency. Toavoid possible inter-sample contamination upon consecutivesample introduction, the capillary inlet was mechanicallyground into a tapered tip with an outer diameter of about70 μm.The tapered tip decreased the contact area betweenthe capillary and droplet. Thus, the amount of residual de-creased accordingly. Moreover, inter-sample contaminationcan be further reduced by using covering oil. This is due tothe washing and brushing effect when the capillary is vertical-ly removed from the chip. The results of consecutive separa-tion showed that the status of all genes from various sampleswas accurately identified according to the peak pattern in theelectrophoretogram. No additional peaks were observed dueto inter-sample contamination. Thus, no washing step wasneeded between samples for consecutive detection with thepresent system, significantly shortening the total detectiontime. Here, 30 samples from 10 CRC patients (three genesdetected for each patient) were consecutively introduced andseparated by droplet-based CE under an automatic controlmodule, and their mutations were detectable with excellentaccuracy and sensitivity.

It has been shown that mutant KRAS, BRAF, and PIK3CAare associated with poor outcome in anti-EGFR-targeted ther-apy [42, 43]. We therefore detected their gene status in CRCpatient tissues using the present system. Existing literaturedemonstrates that mutant KRAS and BRAF may not coexist

Table 1 The genemutation status in 10clinical samples detectedby droplet-based CEsystem

Sample KRAS BRAF PIK3CA

1 M* W M*

2 W W W

3 M W W

4 W W M*

5 M W W

6 M W M

7 W M M

8 W W W

9 M W M*

10 M* W W

W, wild type; M, mutant type; *The muta-tion confirmed by clone sequencing

Consecutive and automatic detection of multi-gene mutations from colorectal cancer samples by coupling... 3045

in CRC patients, but KRAS and PIK3CA or BRAF andPIK3CAwere found to do so [44, 45]. Our study showed thatspecimens 1 and 9 carried both mutant KRAS and PIK3CA,and specimen 7 harboured both mutant BRAF and PIK3CA.However, we did not observe the coexistence of KRAS andBRAF mutations in any detected specimens. This is in agree-ment with existing reports regarding the correlation between

the three genes and mutation status. This also indicated thenecessity of multi-gene mutation detection for guidingtargeted therapy.

Among 30 detected samples from 10 CRC patients, 12mutations were detectable by the droplet-based CE system.Seven of these were confirmed by Sanger sequencing, butothers had to be identified via more sensitive clone

Fig. 5 Consecutive detection of KRAS, BRAF, and PIK3CA mutation in10 CRC FFPE samples using droplet-based CE system. The upper panelshows the electropherogram of 30-sample consecutive genotyping from

10 specimens. The lower panel shows the enlarged electropherograms ofindividual analysis for all samples. The mutant peaks discovered by thedroplet-based CE system are indicated with arrows

Feng Y. et al.3046

sequencing. This demonstrated the excellent sensitivity andaccuracy of the system. Notably, specimen 4 only carried thePIK3CA mutation with a mutation frequency of 0.42%, andspecimen 10 only carried the KRAS mutation with a mutationfrequency of less than 0.37%. Both mutation rates are largelybeyond the detection limit of Sanger sequencing and NGS,suggesting that if a targeted therapeutic strategy were madebased on routine sequencing methods, patients could poten-tially be incorrectly classified. In spite of limited amounts ofthe specimens being detected, the results demonstrated thatour system had the ability to consecutively detect multi-genemutations with high accuracy and sensitivity, suggesting it tobe a promising tool for guiding targeted therapy.

Conclusion

We successfully detected multi-gene mutations from CRC pa-tients via coupling droplet array-based CE and PCR-RFLPtechnology. Small volumes of DNA samples (nanolitre-scaleinitial volume) could be detected with high sensitivity by thedroplet-based electrokinetic sample introduction method.Multi-samples were consecutively analysed by a “∩” shapecapillary probe-based vertical sample introduction method un-der an automatic control system. Due to the effective connec-tion between droplets and CE, the present system has the po-tential to integrate CE separation with droplet-based PCR, evenwith droplet in situ cell lysis and DNA extraction steps. In thisstudy, only 200 nL of initial sample volume was needed forsensitive CE separation, which would facilitate the connectionto the upstream assays such as PCR and digestion in droplet.After the integration, only nanolitre-scale sample is requiredinstead of using the microlitre-scale sample in this study. Thiscan potentially lead to continuous and automatic control of thefull process for gene mutation detection. Further, the systemcould also be used to automatically analyse non-invasive bloodsamples with high efficacy via integration, serving as a moreeffective platform for individualised therapy.

The current issue with the droplet-based system is that thesieving matrix injection process was performed by taking thecapillary out the CE system, which is labour-intensive andpotentially causing the capillary to deviate from the focus ofthe laser beam. Therefore, we are planning to develop an in-tegrated gel injection module which can pump the matrix tothe capillary in the CE instrument.

Funding information This research work was financially supported bythe National Natural Science Foundation of China (Grant number81672920 and 21375149).

Compliance with ethical standards The ethics committeeapproved the use of these samples for the purpose of academic research,and all patients who donated samples provided written informed consent.

Conflict of interest The authors declare that they have no conflict ofinterest.

References

1. Suzawa K, Offin M, Lu D, Kurzatkowski C, Vojnic M, Smith RS,et al. Activation of KRASmediates resistance to targeted therapy inMET exon 14-mutant non-small cell lung cancer. Clin Cancer Res.2019;25:1248–60.

2. Piawah S, Venook AP. Targeted therapy for colorectal cancer me-tastases. A review of current methods of molecularly targeted ther-apy and the use of tumor biomarkers in the treatment of metastaticcolorectal cancer. Cancer. 2019;125:4139–47.

3. Sorich MJ, Wiese MD, Rowland A, Kichenadasse G, McKinnonRA, Karapetis CS. Extended RASmutations and anti-EGFRmono-clonal antibody survival benefit in metastatic colorectal cancer: ameta-analysis of randomized, controlled trials. Ann Oncol.2015;26:13–21.

4. Santos C, Azuara D, Garcia-Carbonero R, Alfonso PG, Carrato A,Elez ME, et al. Optimization of RAS/BRAF mutational analysisconfirms improvement in patient selection for clinical benefit toanti-EGFR treatment in metastatic colorectal cancer. Mol CancerTher. 2017;16:1999–2007.

5. Aleksakhina SN, Kashyap A, Imyanitov EN. Mechanisms of ac-quired tumor drug resistance. Biochim Biophys Acta Rev Cancer.2019;1872:188310.

6. Ramos P, Bentires-Alj M. Mechanism-based cancer therapy: resis-tance to therapy, therapy for resistance. Oncogene. 2015;34:3617–26.

7. Rotow JK, Gui P, Wu W, Raymond VM, Lanman RB, Kaye FJ,et al. Co-occurring alterations in the RAS-MAPK pathway limitresponse to MET inhibitor treatment in MET exon 14 skippingmutation-positive lung cancer. Clin Cancer Res. 2020;26:439–49.

8. Zhao B, Wang L, Qiu H, Zhang M, Sun L, Peng P, et al.Mechanisms of resistance to anti-EGFR therapy in colorectal can-cer. Oncotarget. 2017;8:3980–4000.

9. Yaeger R, Kotani D, Mondaca S, Parikh AR, Bando H, VanSeventer EE, et al. Response to anti-EGFR therapy in patients withBRAF non-V600-mutant metastatic colorectal cancer. Clin CancerRes. 2019;25:7089–97.

10. Tural D, Batur S, Erdamar S, Akar E, Kepil N, Mandel NM, et al.Analysis of PTEN, BRAF and PI3K status for determination ofbenefit from cetuximab therapy in metastatic colorectal cancer pa-tients refractory to chemotherapy with wild-type KRAS. TumourBiol. 2014;35:1041–9.

11. Kwon Y, KimM, Jung HS, Kim Y, Jeoung D. Targeting autophagyfor overcoming resistance to anti-EGFR treatments. Cancers(Basel). 2019;11:1374.

12. French D, Smith A, Powers MP, Wu AHB. KRAS mutation detec-tion in colorectal cancer by a commercially available gene chiparray compares well with Sanger sequencing. Clin Chim Acta.2011;412:1578–81.

13. Del Vecchio F, Mastroiaco V, Di Marco A, Compagnoni C, CapeceD, Zazzeroni F, et al. Next-generation sequencing: recent applica-tions to the analysis of colorectal cancer. J Transl Med. 2017;15:1–19.

14. Spencer DH, Zhang B, Pfeifer J. Single nucleotide variant detectionusing next generation sequencing. 1st ed: Elsevier; 2015. p. 109–27.

15. Kakavas VK, Konstantinos KV, Plageras P, Panagiotis P, VlachosTA, Antonios VT, et al. PCR-SSCP: a method for the molecularanalysis of genetic diseases. Mol Biotechnol. 2008;38:155–63.

16. Liu W, Li B, Chu H, Zhang Z, Luo L, Ma W, et al. Rapid detectionof mutations in erm (41) and rrl associated with clarithromycin

Consecutive and automatic detection of multi-gene mutations from colorectal cancer samples by coupling... 3047

resistance inMycobacterium abscessus complex by denaturing gra-dient gel electrophoresis. J Microbiol Methods. 2017;143:87–93.

17. Li WM, Hu TT, Zhou LL, Feng YM, Wang YY, Fang J. Highlysensitive detection of the PIK3CA H1047R mutation in colorectalcancer using a novel PCR-RFLP method. BMC Cancer. 2016;16:1–11.

18. Lang AH, Drexel H, Geller-Rhomberg S, Stark N, Winder T,Geiger K, et al. Optimized allele-specific real-time PCR assaysfor the detection of common mutations in KRAS and BRAF. JMol Diagn. 2011;13:23–8.

19. Huang Y, Wei L, Sun AM, Li B, Sun CJ, Liang WB, et al.Application of multiplex methylated-specific PCR with capillaryelectrophoresis to explore prognostic value of TSGs hypermethy-lation for hepatocellular carcinoma. J Clin Lab Anal. 2018;32:e22430.

20. Ruan J, Li M, Liu YP, Li YQ, Li YX. Rapid and sensitive detectionof Cronobacter spp. (previously Enterobacter sakazakii) in food byduplex PCR combined with capillary electrophoresis-laser-inducedfluorescence detector. J Chromatogr B Analyt Technol Biomed LifeSci. 2013;921–2:15–20.

21. Harstad RK, Johnson AC, Weisenberger MM, Bowser MT.Capillary electrophoresis. Anal Chem. 2016;88:299–319.

22. Wang R, Xie H, Xu YB, Jia ZP, Meng XD, Zhang JH, et al. Studyon detection of mutation DNA fragment in gastric cancer by restric-tion endonuclease fingerprinting with capillary electrophoresis.Biomed Chromatogr. 2012;26:393–9.

23. Galievsky VA, Stasheuski AS, Krylov SN. “Getting the best sensi-tivity from on-capillary fluorescence detection in capillary electro-phoresis” - a tutorial. Anal Chim Acta. 2016;935:58–81.

24. Price AK, Paegel BM. Discovery in droplets. Anal Chem. 2016;88:339–53.

25. Basova EY, Foret F. Droplet microfluidics in (bio)chemical analy-sis. Analyst. 2015;140:22–38.

26. Niu X, Pereira F, Edel JB, de Mello AJ. Droplet-interfaced micro-chip and capillary electrophoretic separations. Anal Chem.2013;85:8654–60.

27. Zhu Y, Fang Q. Analytical detection techniques for dropletmicrofluidics-a review. Anal Chim Acta. 2013;787:24–35.

28. Li Q, Zhu Y, Zhang NQ, Fang Q. Automatic combination ofmicrofluidic nanoliter-scale droplet array with high-speed capillaryelectrophoresis. Sci Rep. 2016;6:26654.

29. Tan X, Wang H, Luo G, Ren S, Li W, Cui J, et al. Clinical signif-icance of a point mutation in DNA polymerase beta (POLB) gene ingastric cancer. Int J Biol Sci. 2015;11:144–55.

30. FangXX, Li HY, Fang P, Pan JZ, FangQ.A handheld laser-inducedfluorescence detector for multiple applications. Talanta. 2010;150:135–41.

31. Zhang T, Fang Q, DuWB, Fu JL. Microfluidic picoliter-scale trans-lational spontaneous sample introduction for high-speed capillaryelectrophoresis. Anal Chem. 2009;81:3693–8.

32. Ahmed D, Eide PW, Eilertsen IA, Danielsen SA, Eknæs M,Hektoen M, et al. Epigenetic and genetic features of 24 colon can-cer cell lines. Oncogenesis. 2013;2:e71.

33. Rachiglio AM, Lambiase M, Fenizia F, Roma C, Cardone C,Iannaccone A, et al. Genomic profiling of KRAS/NRAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patients revealsnovel mutations in genes potentially associated with resistance toanti-EGFR agents. Cancers (Basel). 2019;11:859.

34. Feinberg AP, Koldobskiy MA, Göndör A. Epigenetic modulators,modifiers and mediators in cancer aetiology and progression. NatRev Genet. 2016;17:284–99.

35. Zhang YX, Zhu Y, Yao B, Fang Q. Nanolitre droplet array for realtime reverse transcription polymerase chain reaction. Lab Chip.2011;11:1545–9.

36. Hatch AC, Ray T, LintecumK, Youngbull C. Continuous flow real-time PCR device using multi-channel fluorescence excitation anddetection. Lab Chip. 2014;14:562–8.

37. Chen F, Rang Y, Weng Y, Lin L, Zeng H, Nakajim H, et al. Drop-by-drop chemical reaction and sample introduction for capillaryelectrophoresis. Analyst. 2015;140:3953–9.

38. Cheng YQ, Yao B, Zhang HD, Fang J, Fang Q. An automatedcapillary electrophoresis system for high-speed separation ofDNA fragments based on a short capillary. Electrophoresis.2010;31:3184–91.

39. Fang XX, Fang P, Pan JZ, Fang Q. A compact short-capillary basedhigh-speed capillary electrophoresis bioanalyzer. Electrophoresis.2016;37:2376–83.

40. Opekar F, Tůma P. Direct sample injection from a syringe needleinto a separation capillary. Anal Chim Acta. 2018;1042:133–40.

41. Shao J, Li S, ZhangW, Fan LY, Cao CX, Sun R, et al. Controlling ofband width, resolution and sample loading by injection system in asimple preparative free-flow electrophoresis with gratis gravity. JChromatogr A. 2010;1217:2182–6.

42. Llovet P, Sastre J, Ortega JS, Bando I, Ferrer M, García-Alfonso P,et al. Prognostic value of BRAF, PI3K, PTEN, EGFR copy number,amphiregulin and epiregulin status in patients with KRAS codon 12wild-type metastatic colorectal cancer receiving first-line chemo-therapy with anti-EGFR therapy. Mol Diagn Ther. 2015;19:397–408.

43. Pietrantonio F, Vernieri C, Siravegna G, Mennitto A, Berenato R,Perrone F, et al. Heterogeneity of acquired resistance to anti-EGFRmonoclonal antibodies in patients with metastatic colorectal cancer.Clin Cancer Res. 2017;23:2414–22.

44. Garcia-Albeniz X, Pericay C, Alonso-Espinaco V, Alonso V,Escudero P, Fernández-Martos C, et al. Serum matrilysin correlateswith poor survival independently of KRAS and BRAF status inrefractory advanced colorectal cancer patients treated withirinotecan plus cetuximab. Tumour Biol. 2011;32:417–24.

45. Lurkin I, Stoehr R, Hurst CD, van Tilborg AAG, Knowles MA,Hartmann A, et al. Two multiplex assays that simultaneously iden-tify 22 possible mutation sites in the KRAS, BRAF, NRAS andPIK3CA genes. PLoS One. 2010;5:e8802.

Publisher’s note Springer Nature remains neutral with regard to jurisdic-tional claims in published maps and institutional affiliations.

Yiming Feng received his Ph.D.degree in cell biology from theChina Medical University. His re-search interest was focused oncapillary electrophoresis andmicrofluidics. Now, he is workingin a Gene Technologies companyas Senior Engineer. His work fo-cuses on the development of inte-grated CE-based medical instru-ment.

Feng Y. et al.3048

Tingting Hu received her mas-ter’s degree in cell biology fromChinaMedical University. Her re-search interest was focused on thecapillary electrophoresis and genemutation detection. Now, she isworking in Ansteel GeneralHospital in the field of immuno-therapy and gene detection forcancer diagnostics.

Pan Fang received her Ph.D. de-gree in chemistry from ZhejiangUniversity. Her research interestw a s f o c u s e d o n t h eminiaturisation of high-speedcapillary electrophoresis and thecombination of capillary electro-phoresis and mass spectrometry.Currently, she is working on anew drug development withsingle-cell droplet screening andmonoclonal antibody characteri-sation using multiple analysistechniques.

Linlin Zhou received her master’sdegree in cell biology from ChinaMedical University. Her researchinterest was focused on the iden-tification of tumour markers.Now, she is a research assistantin the institute of immunotherapyof Fujian Medical University. Herresearch interest is focused on theflow and mass cytometry in thefield of cancer immunology.

Wanming L i i s Assoc ia t eProfessor in the Cell BiologyDepartment at China MedicalUniversity. Her main research in-terest is focused on the identifica-tion of tumour markers and theestablishment of efficient detec-tion methods (metastatic colorec-tal cancer, triple negative breastcancer, poorly differentiated gas-tric cancer, etc.).

Q u n F a n g i s Q i u s h iDistinguished Professor in theDepartment of Chemistry atZhejiang University and Directorof the Institute of MicroanalyticalSystems in the Department ofChemistry. He received his Ph.D.in pharmaceutical analysis fromShenyang Pharmaceu t i ca lUniversity in 1998. His researchinterests include microfluidicanalysis, capillary electrophore-sis, liquid chromatography, massspectrometry, and miniaturisationof analytical instruments, espe-

cially the development of automated and high-throughput droplet-basedmicrofluidic analysis techniques for high-throughput screening, single-cell analysis, biochemical analysis, and point-of-care testing.

Jin Fang is Professor in theDepartment of Cell Biology atChinaMedical University. Her re-search interest is focused on thediscovery and identification ofcancer biomarkers, and also ondevelopment of microfluidics-based techniques to detect the bio-markers for cancer early diagnosisand therapy monitoring.

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